Animal-free dietary collagen

ABSTRACT

Provided herein are non-naturally occurring polypeptides comprising a sequence of a fragment of a collagen and recombinant cells containing heterologous nucleic acid sequences encoding the non-naturally occurring polypeptides. Further provided herein are animal-free methods of generating and purifying such non-naturally occurring polypeptides using microorganisms, preferably from bacterial cells.

CROSS-REFERENCE

This application is a continuation application of U.S. patentapplication Ser. No. 17/171,874, filed Feb. 9, 2021, which is acontinuation application of International Patent Application No.PCT/US2021/014714, filed Jan. 22, 2021, which application claims thebenefit of U.S. Provisional Application Nos. 62/965,700, filed Jan. 24,2020, and 63/117,243, filed Nov. 23, 2020, each of which areincorporated herein by reference in their entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Sep. 27, 2021, isnamed 57607_708_302_SL.txt and is 71,475 bytes in size.

BACKGROUND

Collagen is one of the most abundant proteins found in variousconnective tissues in the body including tendons, ligaments, skin, andhair. Collagens or collagen supplements are popular in medical,cosmetic, and/or health purposes (e.g., stimulating skin growth,promoting wound healing, strengthening nails or joints, etc.). Collagensfor most collagen supplements are derived from animals as a byproduct ofthe animal processing industry. Yet, such animal-derived collagens mayincrease the risk of illness transmission as well as allergies.Moreover, certain consumers are generally interested in animal-freeproducts for a variety of other reasons. Thus, there remains a need forimproved compositions and methods of collagens derived from non-animalsources.

SUMMARY

In one aspect, a non-naturally occurring polypeptide is providedcomprising an amino acid sequence having: (i) at least 80% sequenceidentity to SEQ ID NO: 31 with an N-terminal truncation, a C-terminaltruncation, or both; or (ii) at least 80% sequence identity to SEQ IDNO: 32 with an N-terminal truncation, a C-terminal truncation, or both.In some cases, the non-naturally occurring polypeptide comprises anamino acid sequence having: (i) at least 85% sequence identity to SEQ IDNO: 31 with an N-terminal truncation, a C-terminal truncation, or both;or (ii) at least 85% sequence identity to SEQ ID NO: 32 with anN-terminal truncation, a C-terminal truncation, or both. In some cases,the non-naturally occurring polypeptide comprises an amino acid sequencehaving: (i) at least 90% sequence identity to SEQ ID NO: 31 with anN-terminal truncation, a C-terminal truncation, or both; or (ii) atleast 90% sequence identity to SEQ ID NO: 32 with an N-terminaltruncation, a C-terminal truncation, or both. In some cases, thenon-naturally occurring polypeptide comprises an amino acid sequencehaving: (i) at least 95% sequence identity to SEQ ID NO: 31 with anN-terminal truncation, a C-terminal truncation, or both; or (ii) atleast 95% sequence identity to SEQ ID NO: 32 with an N-terminaltruncation, a C-terminal truncation, or both. In some cases, thenon-naturally occurring polypeptide comprises an amino acid sequencehaving: (i) at least 98% sequence identity to SEQ ID NO: 31 with anN-terminal truncation, a C-terminal truncation, or both; or (ii) atleast 98% sequence identity to SEQ ID NO: 32 with an N-terminaltruncation, a C-terminal truncation, or both. In some cases, thenon-naturally occurring polypeptide comprises: (i) the amino acidsequence of SEQ ID NO: 31 with an N-terminal truncation, a C-terminaltruncation, or both; or (ii) the amino acid sequence of SEQ ID NO: 32with an N-terminal truncation, a C-terminal truncation, or both. In somecases, the non-naturally occurring polypeptide comprises an amino acidsequence having at least 80% sequence identity to SEQ ID NO: 31 with anN-terminal truncation. In some cases, the N-terminal truncation is anN-terminal truncation of 50 amino acids to 600 amino acids. In somecases, the non-naturally occurring polypeptide comprises an amino acidsequence having at least 80% sequence identity to SEQ ID NO: 31 with aC-terminal truncation. In some cases, the C-terminal truncation is aC-terminal truncation of 50 amino acids to 250 amino acids. In somecases, the non-naturally occurring polypeptide comprises an amino acidsequence having at least 80% sequence identity to SEQ ID NO: 31 withboth an N-terminal truncation and a C-terminal truncation. In somecases, the N-terminal truncation is an N-terminal truncation of 50 aminoacids to 600 amino acids, and the C-terminal truncation is a C-terminaltruncation of 50 amino acids to 250 amino acids. In some cases, thenon-naturally occurring polypeptide comprises the amino acid sequence ofSEQ ID NO: 2 or SEQ ID NO: 6. In some cases, the non-naturally occurringpolypeptide consists of the amino acid sequence of SEQ ID NO: 2 or SEQID NO: 6. In some cases, the non-naturally occurring polypeptidecomprises an amino acid sequence having at least 80% sequence identityto SEQ ID NO: 32 with an N-terminal truncation. In some cases, theN-terminal truncation is an N-terminal truncation of 50 amino acids to750 amino acids. In some cases, the non-naturally occurring polypeptidecomprises an amino acid sequence having at least 80% sequence identityto SEQ ID NO: 32 with a C-terminal truncation. In some cases, theC-terminal truncation is a C-terminal truncation of 50 amino acids to250 amino acids. In some cases, the non-naturally occurring polypeptidecomprises an amino acid sequence having at least 80% sequence identityto SEQ ID NO: 32 with both an N-terminal truncation and a C-terminaltruncation. In some cases, the N-terminal truncation is an N-terminaltruncation of 50 amino acids to 750 amino acids, and the C-terminaltruncation is a C-terminal truncation of 50 amino acids to 250 aminoacids. In some cases, the non-naturally occurring polypeptide comprisesthe amino acid sequence of SEQ ID NO: 8. In some cases, thenon-naturally occurring polypeptide consists of the amino acid sequenceof SEQ ID NO: 8. In some cases, the non-naturally occurring polypeptidehas a total truncation of 50 amino acids to 900 amino acids. In somecases, the non-naturally occurring polypeptide is 50 amino acids to 250amino acids in length. In some cases, the non-naturally occurringpolypeptide does not comprise one or more of: a laminin G domain, a VonWillebrand factor type A (vWA) domain, and a fibrillar collagenC-terminal domain. In some cases, the non-naturally occurringpolypeptide comprises one or more collagen triple helix repeats. In somecases, the non-naturally occurring polypeptide is monomeric. In somecases, the non-naturally occurring polypeptide does not form a stabletriple helix structure of a naturally occurring collagen. In some cases,the non-naturally occurring polypeptide is substantially free of othercollagen chains. In some cases, the non-naturally occurring polypeptidehas a non-naturally occurring level of hydroxylation relative to anaturally-occurring collagen. In some cases, fewer than 10% of prolinespresent in the non-naturally occurring polypeptide are hydroxylated. Insome cases, the non-naturally occurring polypeptide is non-hydroxylated.In some cases, the non-naturally occurring polypeptide has anon-naturally occurring level of glycosylation relative to anaturally-occurring collagen. In some cases, the non-naturally occurringpolypeptide protein comprises less than 5 wt. % glycosylation.

In another aspect, a composition is provided comprising between 0.001%and 30% w/w of the non-naturally occurring polypeptide of any one of thepreceding. In some cases, the composition is formulated for consumptionby an individual. In some cases, the composition is a nutraceutical. Insome cases, the individual is a human.

In another aspect, a method of improving the appearance of the skin, thehair, and/or the nails of a subject, and/or improving bone, muscle,and/or joint health in the subject is provided, the method comprising:administering to the subject a composition of any one of the precedingcompositions. In some cases, the administering comprises orallyadministering to the subject.

In yet another aspect, a recombinant cell is provided containing thereinat least one copy of a heterologous nucleic acid sequence encoding anon-naturally occurring polypeptide of any one of the preceding. In somecases, the recombinant cell is a microbial cell. In some cases, themicrobial cell is a bacterial cell. In some cases, the bacterial cell isof the species Escherichia coli. In some cases, the recombinant celllacks an enzyme that hydroxylates one or more amino acids of thenon-naturally occurring polypeptide. In some cases, the recombinant celllacks prolyl 4-hydroxylase and/or prolyl 3-hydroxylase. In some cases,the heterologous nucleic acid sequence comprises a nucleic acid sequencehaving at least 80% sequence identity to any one of SEQ ID NOs: 1, 3, 5,7, 9, 11, and 25-30. In some cases, the heterologous nucleic acidsequence comprises a nucleic acid sequence having at least 85% sequenceidentity to any one of SEQ ID NOs: 1, 3, 5, 7, 9, 11, and 25-30. In somecases, the heterologous nucleic acid sequence comprises a nucleic acidsequence having at least 90% sequence identity to any one of SEQ ID NOs:1, 3, 5, 7, 9, 11, and 25-30. In some cases, the heterologous nucleicacid sequence comprises a nucleic acid sequence having at least 95%sequence identity to any one of SEQ ID NOs: 1, 3, 5, 7, 9, 11, and25-30. In some cases, the heterologous nucleic acid sequence comprises anucleic acid sequence having at least 98% sequence identity to any oneof SEQ ID NOs: 1, 3, 5, 7, 9, 11, and 25-30. In some cases, thenon-naturally occurring polypeptide further comprises a secretionsignal. In some cases, the recombinant cell secretes the non-naturallyoccurring polypeptide into the periplasm, into a culture media, orextracellularly. In some cases, the heterologous nucleic acid sequenceis codon-optimized for expression in the recombinant cell. In somecases, the heterologous nucleic acid sequence is operably linked to aninducible promoter or a constitutive promoter. In some cases, theheterologous nucleic acid is or is contained in a plasmid. In somecases, the heterologous nucleic acid sequence is stably integrated intoa chromosome of the recombinant cell.

In yet another aspect, a culture medium is provided comprising arecombinant cell of any one of the preceding. In some cases, the culturemedium further comprises the non-naturally occurring polypeptide of anyone of the preceding secreted from the recombinant cell.

The present disclosure further provides a recombinant cell containingtherein at least one copy of a heterologous nucleic acid sequenceencoding collagen selected from the group consisting of: Gallus galluscollagen or Acipenser schrenckii (Japanese sturgeon) collagen. In someembodiments, the recombinant cell is a microbial cell. In someembodiments, the microbial cell is a bacterial cell. In someembodiments, the bacterial cell is of the species Escherichia coli. Insome embodiments, the heterologous nucleic acid sequence comprises anyone of SEQ ID NOs: 1, 3, 5, 7, 9, 11, and 25-30.

In some embodiments, the collagen is a Gallus gallus Type 21 collagen.In some embodiments, the collagen is a Acipenser schrenckii Type 2 alpha1 collagen. In some embodiments, the collagen is a non-naturallyoccurring collagen. In some embodiments, the collagen is a truncatedcollagen. In some embodiments, the collagen comprises an amino acidsequence according to any one of SEQ ID NOs: 2, 4, 6, and 8.

In some embodiments, the collagen further comprises a secretion signalsequence. In some instances, the secretion signal sequence comprises anamino acid sequence according to any one of SEQ ID NOs: 10, 12, 14, 16,18, 20, 22, and 24. In some embodiments, the recombinant cell secretesthe collagen into a culture media. In some embodiments, the recombinantcell secretes to the periplasm. In some embodiments, the recombinantcell secrets the collagen to the extracellular space.

In some embodiments, the heterologous nucleic acid sequence iscodon-optimized for expression in the recombinant cell. In someembodiments, the heterologous nucleic acid sequence is operably linkedto an inducible promoter or a constitutive promoter. In someembodiments, the heterologous nucleic acid is or is contained within aplasmid. In some embodiments, the heterologous nucleic acid sequence isstably integrated into the chromosome of the recombinant cell.

The present disclosure also provides a culture medium comprising arecombinant cell described herein. In some embodiments, the culturemedium further comprises a recombinant collagen secreted from therecombinant cell.

The present disclosure also provides a recombinant protein comprising asequence that has at least 90% sequence identity to a fragment of acollagen selected from the group consisting of: Gallus gallus collagen,and Acipenser schrenckii collagen. In some embodiments, the collagen isa Gallus gallus Type 21 collagen. In some embodiments, the collagen is aAcipenser schrenckii Type 2 alpha 1 collagen.

In some embodiments, the collagen is a non-naturally occurring collagenor fragment thereof. In some embodiments, the protein has anon-naturally occurring level of glycosylation (e.g., relative to acorresponding natural collagen). In some embodiments, the proteincomprises less than 5 wt. % glycosylation (e.g., less than 3 wt. %, lessthan 1 wt. %, less than 0.5 wt. %, or less than 0.1 wt. %). In someembodiments, the protein is a truncated collagen. In some embodiments,the protein comprises an amino acid sequence according to any one of SEQID NOs: 2, 4, 6, and 8 (or having a sequence identity of at least 90%thereof, at least 95% thereof, at least 98% thereof, or the like).

In some embodiments, the collagen further comprises a secretion signalsequence. In some embodiments, the secretion signal sequence comprisesan amino acid sequence according to SEQ ID NO: 10, 12, 14, 16, 18, 20,22, and 24.

The present disclosure also provides a composition comprising arecombinant protein as disclosed herein. In some embodiments, thecomposition further comprises a culture media. Additionally and/oralternatively, the composition further comprises a recombinant cell asdisclosed herein. In some embodiments, the recombinant cell is amicrobial cell. In some embodiments, the microbial cell is a bacterialcell. In some embodiments, the bacterial cell is of the speciesEscherichia coli. In some embodiments, the recombinant cell comprises anintegrated heterologous nucleic acid sequence encoding a collagen, atruncated collagen, or fragment thereof. In some embodiments, theheterologous nucleic acid sequence comprises any one of SEQ ID NOs: 1,3, 5, 7, and 25-30.

The present disclosure also provides a process for purifying arecombinant collagen, the process comprises incubating a recombinantcell described herein in a culture media wherein the recombinant cellsecretes the recombinant collagen into the culture media, collecting theculture media comprising the recombinant collagen secreted thereto, andpurifying the recombinant collagen from the culture media.

The present disclosure also provides a recombinant collagen purifiedfrom the culture medium disclosed in the process herein. In someembodiments, the recombinant collagen has a purity of at least 80%, atleast 85%, at least 90%, at least 95%, or at least 99%.

The present disclosure also provides an expression vector comprising anucleic acid sequence encoding a non-naturally occurring truncatedcollagen operably linked to a promoter, wherein the non-naturallyoccurring truncated collagen is selected from the group consisting of:Gallus gallus collagen and Acipenser schrenckii collagen. In someembodiments, the nucleic acid sequence comprises any one of SEQ ID NOs:1, 3, 5, 7, and 25-30. In some embodiments, the Gallus gallus collagenis Type 21 collagen. In some embodiments, the Acipenser schrenckiicollagen is Type 2 alpha 1 collagen.

In some embodiments, the expression vector further comprises a nucleicacid sequence encoding a secretion signal sequence. In some embodiments,the nucleic acid sequence encoding the secretion signal sequencecomprises any one of SEQ ID NOs: 11, 13, 15, 17, 19, 21, and 23. In someembodiments, the nucleic acid sequence is codon optimized for expressionin a cell.

The present disclosure also provides a composition comprising arecombinant collagen disclosed herein, formulated for consumption by anindividual. In some embodiments, the composition is a nutraceutical. Insome embodiments, the individual is a human. In some embodiments, thecomposition comprises from 0.1% to 10% recombinant collagen. In someembodiments, the composition comprises at least 50% of recombinantcollagen. In some embodiments, the composition comprises from 70% to 99%of recombinant collagen. In some embodiments, the composition furthercomprises at least one of a carrier and a preservative.

The present disclosure also provides a method of improving theappearance of the skin, the hair, and/or the nails of a subject byadministering to a subject the composition disclosed herein. In someembodiments, the step of administering comprises orally administering tothe subject.

Additional aspects and advantages of the present disclosure will becomereadily apparent to those skilled in this art from the followingdetailed description, wherein only illustrative embodiments of thepresent disclosure are shown and described. As will be realized, thepresent disclosure is capable of other and different embodiments, andits several details are capable of modifications in various obviousrespects, all without departing from the disclosure. Accordingly, thedrawings and description are to be regarded as illustrative in nature,and not as restrictive.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the subject matter disclosed herein are set forthwith particularity in the appended claims. A better understanding of thefeatures and advantages of the subject matter disclosed herein will beobtained by reference to the following detailed description that setsforth illustrative embodiments, in which the principles of the subjectmatter disclosed herein are utilized, and the accompanying drawings ofwhich:

FIG. 1 shows an image of two SDS-PAGE gels showing bands of collagenproteins in supernatant samples from microbial cell cultures. Theidentities of each protein are indicated above each band.

FIGS. 2A-2C depict images of SDS-PAGE gels showing bands ofnon-naturally occurring polypeptides of the disclosure before and afterpH 3.0 treatment.

FIG. 3 depicts increased cell viability of human dermal fibroblasts whentreated with a non-naturally occurring polypeptide of the disclosure(comprising an amino acid sequence according to SEQ ID NO: 2).

FIG. 4 depicts increased collagen type I production in human dermalfibroblasts when treated with a non-naturally occurring polypeptide ofthe disclosure (comprising an amino acid sequence according to SEQ IDNO: 2).

FIG. 5 depicts increased collagen type I production in tenocytes whentreated with a non-naturally occurring polypeptide of the disclosure(comprising an amino acid sequence according to SEQ ID NO: 2).

FIG. 6 depicts alignments of non-naturally occurring polypeptides of thedisclosure with corresponding naturally occurring collagens. FIG. 6discloses SEQ ID NOS: 33 and 34, respectively, in order of appearance.

FIG. 7A depicts the effect of pH on viscosity of a solution of anexemplary non-naturally occurring polypeptide of the disclosure.

FIG. 7B depicts a comparison of the viscosity of a solution of anexemplary non-naturally occurring polypeptide of the disclosure versus abenchmark.

FIG. 8 depicts viscosity of various blends of an exemplary non-naturallyoccurring polypeptide of the disclosure and xanthan.

FIG. 9 depicts gel hardness of a solution of an exemplary non-naturallyoccurring polypeptide of the disclosure.

FIG. 10A and FIG. 10B depict gel hardness of solutions of various lotsof an exemplary non-naturally occurring polypeptide of the disclosure.

FIG. 11 depicts the effect of compaction and lecithin agglomeration onan exemplary non-naturally occurring polypeptide of the disclosure.

FIG. 12 depicts the effect of pH and oil type on gel hardness of gelscontaining an exemplary non-naturally occurring polypeptide of thedisclosure.

DETAILED DESCRIPTION Definitions

The terminology used herein is for the purpose of describing particularcases only and is not intended to be limiting. As used herein, thesingular forms “a”, “an”, and “the” are intended to include the pluralforms as well, unless the context clearly indicates otherwise.Furthermore, to the extent that the terms “including”, “includes”,“having”, “has”, “with”, or variants thereof are used in either thedetailed description and/or the claims, such terms are intended to beinclusive in a manner similar to the term “comprising”.

The terms “about” or “approximately” mean within an acceptable errorrange for the particular value as determined by one of ordinary skill inthe art, which will depend in part on how the value is measured ordetermined, e.g., the limitations of the measurement system. Forexample, “about” can mean within 1 or more than 1 standard deviation,per the practice in the given value. Where particular values aredescribed in the application and claims, unless otherwise stated theterm “about” should be assumed to mean an acceptable error range for theparticular value.

The terms “individual”, “patient”, or “subject” are used interchangeablyherein. None of the terms require or are limited to a situationcharacterized by the supervision (e.g., constant or intermittent) of ahealth care worker (e.g., a doctor, a registered nurse, a nursepractitioner, a physician's assistant, an orderly, or a hospice worker).

As used herein, the term “comprise” or variations thereof such as“comprises” or “comprising” are to be read to indicate the inclusion ofany recited feature but not the exclusion of any other features. Thus,as used herein, the term “comprising” is inclusive and does not excludeadditional, unrecited features. In some embodiments of any of thecompositions and methods provided herein, “comprising” may be replacedwith “consisting essentially of” or “consisting of”. The phrase“consisting essentially of” is used herein to require the specifiedfeature(s) as well as those which do not materially affect the characteror function of the claimed disclosure. As used herein, the term“consisting” is used to indicate the presence of the recited featurealone.

Throughout this disclosure, various embodiments are presented in a rangeformat. It should be understood that the description in range format ismerely for convenience and brevity and should not be construed as aninflexible limitation on the scope of any embodiments. Accordingly, thedescription of a range should be considered to have specificallydisclosed all the possible subranges as well as any individual numericalvalues within that range to the tenth of the unit of the lower limitunless the context clearly dictates otherwise. For example, descriptionof a range such as from 1 to 6 should be considered to have specificallydisclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from2 to 4, from 2 to 6, from 3 to 6, etc., as well as any individual valueswithin that range, for example, 1.1, 2, 2.3, 5, and 5.9. This appliesregardless of the breadth of the range. The upper and lower limits ofthese intervening ranges may independently be included in the smallerranges, and are also encompassed within the disclosure, subject to anyspecifically excluded limit in the stated range. Where the stated rangeincludes one or both of the limits, ranges excluding either or both ofthose included limits are also included in the disclosure, unless thecontext clearly dictates otherwise.

The terms “treatment of”, “treating”, “applying”, “palliating” or“ameliorating” are used herein interchangeably. These terms refer to anapproach for obtaining beneficial or desired results including but notlimited to therapeutic benefit and/or a prophylactic benefit. By“therapeutic benefit” is meant eradication or amelioration of theunderlying disorder being treated. Also, a therapeutic benefit isachieved with the eradication or amelioration of one or more of thephysiological symptoms associated with the underlying disorder such thatan improvement is observed in the patient, notwithstanding that thepatient is still afflicted with the underlying disorder. Forprophylactic benefit, the compositions are, in some embodiments,administered to a patient at risk of developing a particular disease orcondition, or to a patient reporting one or more of the physiologicalsymptoms of a disease, even though a diagnosis of this disease has notbeen made.

The terms “subject”, “individual”, or “patient” are often usedinterchangeably herein. A “subject” can be a biological entitycontaining expressed genetic materials. The biological entity can be aplant, animal, or microorganism, including, for example, bacteria,viruses, fungi, and protozoa. The subject can be tissues, cells andtheir progeny of a biological entity obtained in vivo or cultured invitro. The subject can be a mammal. The mammal can be a human. Thesubject may be diagnosed or suspected of being at high risk for adisease. In some cases, the subject is not necessarily diagnosed orsuspected of being at high risk for the disease.

The term “truncated collagen” as used herein generally refers to apolypeptide that is smaller than a full-length (e.g., natural) collagenwherein one or more portions of the full-length (e.g., natural) collagenis not present. The non-naturally polypeptides provided herein may betruncated at the C-terminal end, the N-terminal end, truncated byremoval of internal portion(s) of the full-length collagen sequence(e.g., an internal truncation), truncated at both the C-terminal end andthe N-terminal end, or may have one or both of a C-terminal truncationand an N-terminal truncation as well as an internal truncation. In anon-limiting embodiment, a truncated collagen may comprise an amino acidsequence according to SEQ ID NO: 2, or a homolog thereof. In anothernon-limiting embodiment, a truncated collagen may comprise an amino acidsequence according to SEQ ID NO: 8, or a homolog thereof.

When used in reference to an amino acid position, a “truncation” isinclusive of said amino acid position. For example, an N-terminaltruncation at amino acid position 100 of a full-length protein means atruncation of 100 amino acids from the N-terminus of the full-lengthprotein (i.e., the truncated protein is missing amino acid positions 1through 100 of the full-length protein). Similarly, a C-terminaltruncation at amino acid position 901 of a full-length protein (assuminga 1000 amino acid full-length protein) means a truncation of 100 aminoacids from the C-terminus (i.e., the truncated protein is missing aminoacid positions 901 through 1000 of the full-length protein). Similarly,an internal truncation at amino acid positions 101 and 200 means aninternal truncation of 100 amino acids of the full-length protein (i.e.,the truncated protein is missing amino acid positions 101 to 200 of thefull-length protein).

The section headings used herein are for organizational purposes onlyand are not to be construed as limiting the subject matter described.

Provided in certain embodiments herein are, by way of non-limitingexample, compositions, methods, and systems for manufacturingnon-naturally occurring polypeptides, such as, e.g., animal-freecollagen polypeptides or collagen-like polypeptides, as well as collagenfragments, and/or truncated collagens, such as that are expressed inand/or by genetically engineered microorganisms. Thus, in variousaspects of the disclosure, the non-naturally occurring polypeptidesprovided herein include collagen or collagen-like polypeptides,recombinant collagens, collagen fragments, or truncated collagens. Incertain embodiments, the non-naturally occurring polypeptides describedherein (e.g., recombinant collagens, collagen fragments, or truncatedcollagens) are derived from any suitable source, such as from mammalianor non-mammalian sources. For example, in some embodiments, thenon-naturally occurring polypeptides described herein (e.g., recombinantcollagens, collagen fragments, or truncated collagens), or at least aportion thereof, are derived from (e.g., modified, truncated, fragmentsof, or the like) collagens of a bird or an avian animal (e.g., Gallusgallus collagen), a freshwater- or saltwater-fish (e.g., Acipenserschrenckii collagen), or any combination thereof.

The non-naturally occurring polypeptides provided herein are notnormally found in nature. Generally, the non-naturally occurringpolypeptides described herein exhibit one or more differences fromnaturally occurring collagens. In certain aspects, the non-naturallyoccurring polypeptides provided herein may have a different amino acidsequence from naturally occurring polypeptides (e.g., a truncatedcollagen). In some cases, the non-naturally occurring polypeptides mayhave a different structure from a naturally occurring collagen. Thequaternary structure of natural collagen is a triple helix, typicallycomposed of three polypeptides. In some aspects, the non-naturallyoccurring polypeptides described herein may not have or may not form aquaternary structure of natural collagen. For example, in someinstances, the non-naturally occurring polypeptides described herein maynot form the stable triple helical structure of naturally occurringcollagen. In certain instances, of the three polypeptides that formnatural collagen, two are usually identical and are designated as thealpha chain. The third polypeptide is designated as the beta chain. Incertain instances, a typical natural collagen can be designated as AAB,wherein the collagen is composed of two alpha (“A”) strands and one beta(“B”) strand. In some aspects, the non-naturally occurring polypeptidesdescribed herein do not have the AAB structure of natural collagen. Insome instances, the non-naturally occurring polypeptides describedherein are free from or substantially free from different collagenchains (e.g., a non-naturally occurring polypeptide described herein maycomprise an alpha chain collagen and may be free or substantially freefrom a beta chain collagen). In some aspects, the non-naturallyoccurring polypeptides described herein are monomeric (e.g., do not formmultimeric structures). In other aspects, the non-naturally occurringpolypeptides described herein may, in some instances, form multimericstructures with identical monomers (e.g., homodimers, homotrimers,etc.).

In some aspects, the non-naturally occurring polypeptides arerecombinant polypeptides (e.g., prepared recombinantly in a host cell).The non-naturally occurring collagen is, in one embodiment, a truncatedcollagen. Other non-naturally occurring collagen polypeptides includechimeric collagens. A chimeric collagen is a polypeptide wherein oneportion of a collagen polypeptide is contiguous with a portion of asecond collagen polypeptide. For example, a collagen molecule comprisinga portion of a collagen from one species contiguous with a portion of acollagen from another species is a chimeric collagen. In anotherembodiment, the non-naturally occurring collagen comprises a fusionpolypeptide that includes additional amino acids such as a secretiontag, histidine tag, green fluorescent protein, protease cleavage site,GEK repeats, GDK repeats, and/or beta-lactamase.

In some embodiments, the non-naturally occurring polypeptides (e.g.,recombinant polypeptides) provided herein have a non-naturally occurringlevel of glycosylation, for example, relative to a corresponding naturalcollagen or naturally present collagen. For example, in someembodiments, the non-naturally occurring polypeptide (e.g., recombinantpolypeptide) comprises less than 10 wt. %, less than 9 wt. %, less than8 wt. %, less than 7 wt. %, less than 6 wt. %, less than 5 wt. %, lessthan 4 wt. %, less than 3 wt. %, less than 2 wt. %, less than 1 wt. %,less than 0.9 wt. %, less than 0.8 wt. %, less than 0.7 wt. %, less than0.6 wt. %, less than 0.5 wt. %, less than 0.4 wt. %, less than 0.3 wt.%, less than 0.2 wt. %, or less than 0.1 wt. % glycosylation.Alternatively and/or additionally, the non-naturally occurringpolypeptide (e.g., recombinant polypeptide) comprises less than 95%,less than 90%, less than 85%, less than 80%, less than 75%, less than70%, less than 65%, less than 60%, less than 55%, less than 50%, lessthan 45%, less than 40%, less than 35%, less than 30%, less than 25%,less than 20%, less than 15%, less than 10%, or less than 5% of totalglycosylation of the corresponding natural collagen or naturally presentcollagen. For example, where the naturally present collagen ABC from aspecies XYZ has 20 glycosylations (throughout the full length of thecollagen ABC or a portion thereof), it is contemplated that thenon-naturally occurring polypeptide (e.g., recombinant polypeptide)comprises less than 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6,5, 4, 3, 2, or 1 glycosylations. In some embodiments, those lower levelsof glycosylation can be specific to one or more types of glycosylation(e.g., O-glycosylation or N-glycosylation, etc.) and/or theglycosylation residues (e.g., galactosylhydroxylysine (Gal-Hyl),glucosyl galactosylhydroxylsine (GlcGal-Hyl), etc.). Non-naturallyoccurring polypeptides produced recombinantly (e.g., in a recombinanthost cell), in some instances, may have a glycosylation level and/or aglycosylation pattern that differs from naturally occurring collagen.

In some aspects, a non-naturally occurring polypeptide provided hereinhas a non-naturally occurring amount of hydroxyprolines. In some cases,a non-naturally occurring polypeptide provided herein lackshydroxyprolines. In some cases, a non-naturally occurring polypeptideprovided herein comprises fewer hydroxyprolines than anaturally-occurring collagen. Hydroxyprolines include, withoutlimitation, 3-hydroxyproline, 4-hydroxyproline, and 5-hydroxyproline. Insome cases, less than about 50% (e.g., less than about 45%, less thanabout 40%, less than about 35%, less than about 30%, less than about25%, less than about 20%, less than about 15%, less than about 10%, orless) of the prolines present in the amino acid sequence of anon-naturally occurring polypeptide provided herein are hydroxyprolines.In some aspects, a non-naturally occurring polypeptide producedrecombinantly (e.g., in a recombinant host cell) may have fewerhydroxyprolines than a naturally occurring collagen. In some cases, arecombinant polypeptide as provided herein is recombinantly expressed ina recombinant host cell (e.g., bacterial cell) that lacks an enzyme thathydroxylates one or more amino acids (e.g., proline) of the recombinantpolypeptide. In some cases, a recombinant polypeptide as provided hereinis recombinantly expressed in a host cell (e.g., bacterial cell) thatlacks prolyl 4-hydroxylase and/or prolyl 3-hydroxylase.

In some aspects, the non-naturally occurring polypeptides providedherein lack or substantially lack lysyl oxidation. Lysyl oxidationinvolves the conversion of lysine residues into highly reactivealdehydes that can form cross-links with other proteins. Naturallyoccurring collagens may have some level of lysyl oxidation. Thus, thenon-naturally occurring polypeptides may be different from naturalcollagens in that they lack or substantially lack lysyl oxidation.

Generally, the non-naturally occurring polypeptides provided herein(e.g., truncated collagens) may have a function and/or provide a benefit(e.g., as provided herein) similar or substantially similar to that of anatural or a full-length collagen. In some cases, the non-naturallyoccurring polypeptides provided herein (e.g., truncated collagens) mayhave improved or increased function and/or benefit (e.g., as providedherein) as compared to a natural or a full-length collagen.

The non-naturally occurring polypeptides disclosed herein often haveadvantageous properties related to their monomeric structure and/or lackof amino acids capable of cross-linking with other collagen strands,e.g., the lack of hydroxyproline residues. In addition, collagenhydrolysates of the non-naturally occurring polypeptides disclosedherein are also produced with increased solubility as compared tofull-length or natural collagens. Moreover, monomeric structures, asopposed to natural triple helix collagens, are more readily digestibleand bioavailable, or broken down by digestive proteases. Otheradvantageous properties include improved physical properties in liquidcompositions and in purification processes, since full-length or naturalcollagens or collagen strands interact to form stronger structures thatcan precipitate due to the presence of hydroxyproline residues.

In certain preferred embodiments, the non-naturally occurringpolypeptides provided herein (e.g., truncated collagens) comprise anamino acid sequence that has at least about 70%, at least about 75%, atleast about 80%, at least about 85%, at least about 90%, at least about91%, at least about 92%, at least about 93%, at least about 94%, atleast about 95%, at least about 96%, at least about 97%, at least about98%, or at least about 99% sequence identity to at least a portion ofthe naturally existing mammalian or non-mammalian collagens from whichthose are derived from. In some instances, a portion or portions of anatural amino acid sequence is deleted, but the remainder of thesequence is substantially similar or identical to the natural amino acidsequence. In certain exemplary embodiments, the non-naturally occurringpolypeptide has an amino acid sequence that has at least about 70%, atleast about 75%, at least about 80%, at least about 85%, at least about90%, at least about 91%, at least about 92%, at least about 93%, atleast about 94%, at least about 95%, at least about 96%, at least about97%, at least about 98%, or at least about 99% sequence identity to aGallus gallus Type 21 alpha 1 collagen or fragment thereof. In anotherexample, the non-naturally occurring polypeptide has an amino acidsequence that has at least about 70%, at least about 75%, at least about80%, at least about 85%, at least about 90%, at least about 91%, atleast about 92%, at least about 93%, at least about 94%, at least about95%, at least about 96%, at least about 97%, at least about 98%, or atleast about 99% sequence identity to a Acipenser schrenckii Type 2 alpha1 collagen fragment.

In some embodiments, the recombinant protein is a truncated collagen. Incertain instances, a truncated collagen is a polypeptide that is smallerthan a full-length (e.g., natural) collagen wherein one or more portions(e.g., internal and/or terminal portion(s)) of the full-length (e.g.,natural) collagen is not present. In various instances, thenon-naturally occurring polypeptides provided herein (e.g., truncatedcollagens) are truncated at the C-terminal end, the N-terminal end,truncated by removal of internal portion(s) of the full-length collagenpolypeptide (e.g., internal truncation), truncated at both theC-terminal end and the N-terminal end, or comprise one or both of aC-terminal truncation and an N-terminal truncation as well as aninternal truncation. In some instances, the non-naturally occurringpolypeptide is a fragment of a naturally occurring collagen that retainsat least 50%, at least 60%, at least 70%, at least 80%, at least 90% ofa function (e.g., of interest) of natural or naturally-presentcorresponding collagens. In some instances, the term truncated collagenis interchangeably used with the term collagen fragment. In someinstances, the truncated collagen includes any contiguous collagenfragments that are at least 10%, at least 20%, at least 30%, at least40%, at least 50%, at least 60%, at least 70%, at least 80% offull-length natural or naturally-present corresponding collagens. Insome embodiments, the truncation is an internal truncation, a truncationat the N-terminal portion of the collagen, a truncation at theC-terminal portion of the collagen, a truncation of an internal portion,or a truncation at both the C-terminal end and the N-terminal end. Atruncated collagen provided herein may be truncated by from 50 aminoacids to 1000 amino acids, from 50 amino acids to 950 amino acids, from50 amino acids to 900 amino acids, from 50 amino acids to 850 aminoacids, from 50 amino acids to 800 amino acids, from 50 amino acids to750 amino acids, from 50 amino acids to 700 amino acids, from 50 aminoacids to 650 amino acids, from 50 amino acids to 600 amino acids, from50 amino acids to 550 amino acids, from 50 amino acids to 500 aminoacids, from 50 amino acids to 450 amino acids, from 50 amino acids to400 amino acids, from 50 amino acids to 350 amino acids, from 50 aminoacids to 300 amino acids, from 50 amino acids to 250 amino acids, from50 amino acids to 200 amino acids, from 50 amino acids to 150 aminoacids, or from 50 amino acids to 100 amino acids. In another embodiment,a truncated collagen is truncated by 50, 60, 70, 80, 90, 100, 110, 120,130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260,270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400,410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540,550, 560, 570, 580, 590, 600, 650, 700, 750, 800, 850, 900, 950, or 1000amino acids.

A non-naturally occurring polypeptide (e.g., truncated collagen)disclosed herein may comprise a truncation relative to a full-length(e.g., natural) collagen. In some embodiments, a truncated collagendisclosed herein may comprise a truncation relative to a full-length(e.g., natural) chicken (Gallus gallus) type 21 alpha 1 collagen (e.g.,SEQ ID NO: 31). In some embodiments, a truncated collagen disclosedherein may comprise the amino acid sequence of SEQ ID NO: 31 with anN-terminal truncation, a C-terminal truncation, an internal truncation,or a combination thereof. In some embodiments, a truncated collagendisclosed herein may comprise a truncation relative to a full-length(e.g., natural) Japanese sturgeon (Acipenser schrenckii) type 2 alpha 1collagen (e.g., SEQ ID NO: 32). In some embodiments, a truncatedcollagen disclosed herein may comprise the amino acid sequence of SEQ IDNO: 32 with an N-terminal truncation, a C-terminal truncation, aninternal truncation, or a combination thereof. Non-limiting examples offull-length (e.g., natural) collagens are provided in Table 1 below.

In other embodiments, polypeptides may be truncated collagenpolypeptides comparable to fish collagens, including from other speciesof sturgeon, or from other species producing roe suitable for caviar,including salmon, steelhead, trout, lumpfish, whitefish, or carp, aswell as other fish such as tilapia and sharks. Suitable comparablesequences from Acipenser schrenckii (Japanese sturgeon) include NCBIaccession numbers BAO58965.1, BAO58966.1, BAO58967.1, BAT51012.1,BAR72360.1, BAR72359.1, BAR72358.1, BAR72357.1 and BAR72356.1. Suitablesequences from Acipenser ruthemus (Sterlet sturgeon) include NCBIaccession numbers A0A444UGW0, A0A444TZM6, A0A444UC45, A0A444UC53,A0A662YTX1, A0A662Z270, A0A662YZ39, A0A444U1F5, A0A444UJK3, A0A444UNU0,X5HZZ7, X5IHC1, A0A444UPK8, A0A444UBS1, A0A444UYQ7, A0A444TWQ3,A0A444ULY4, A0A444TZ23, A0A662YS48, A0A444U4C8, A0A444UD64, A0A662YX10,A0A662YXI2, A0A444TXQ4, A0A444TZ42, A0A444U8N8, A0A444UJU3, A0A444UQ51,A0A444U2T2, A0A662YJ50, A0A444V1V9, A0A444V113, A0A662YWR6, A0A662YW91,A0A444U5J5, A0A662YR93, A0A444UJB0, A0A444UFS4, A0A444UVK2, A0A444UJU1,A0A444ULY9, A0A444UKA7, A0A444U5L7, A0A444V6M4, A0A444V788, A0A444UFS9,A0A444UVP7, A0A444U4D9, A0A444UHN6, A0A662YJC1, A0A444V1E8, A0A444UPM0,A0A662YU87, A0A444TZS8, A0A444U200, A0A444V2E3, A0A662YXD3, A0A662YQA4,A0A444U1H9, A0A444V715, A0A444UFX8, A0A444V7B8, A0A444U2K4, A0A444V762,A0A444UQ49, A0A662YMD3, A0A662YWF2, A0A444UE44, A0A444UAR6, A0A444UX46,A0A444U5P4, A0A662YRG8, A0A444USC3, A0A444UK09, A0A444UNQ7, A0A444UN69,A0A444V5D9, E6Y298, A0A444TZY1, A0A444TYS0, and E6Y299.

In other embodiments, polypeptides may be truncated collagenpolypeptides comparable to chicken collagens, or other poultrycollagens, such as from domestic fowls, including chickens, turkeys,geese, and ducks. Suitable comparable sequences from Gallus gallus(chicken) include NCBI accession numbers V9GZR2, Q9PSS5, A0A3Q2UDI3,Q90802, A0A1D5PNH7, Q4TZW6, Q90803, Q91014, A0A1D5PPI0, A0A1D5P1A5,A0A3Q2U6K2, A0A3Q2U8F9, Q90689, A0A3Q2U3U6, P13731, A0A1D5PFE0,A0A3Q2TXZ7, Q5FY72, A0A1D5PR16, A0A1D5PKR6, F1NDF5, Q90589, P08125,F1NRH2, P32017, A0A1D5PW49, Q90800, P12108, E1C353, Q7LZR2, P02460,A0A1L1RNI7, Q90796, P12106, F1NQ20, Q919K3, P20785, A0A1D5PWN6, P15988,P12105, F1NIL4, 093419, P02467, A0A5H1ZRJ7, A0A1D5PKQ4, A0A5H1ZRK9,Q90W37, A0A1D5NY11, A0A1D5P959, P02457, A0A1D5PYU1, A0A1D5PE57, Q9OZA0,Q90584, A0A1L1RZW7, A0A1D5NVM0, A0A1D5P8P3, F1NIP0, F1P2Q3, A0A1D5PE74,Q9IAU4, A0A3Q2TTC1, F1NHH4, P32018, A0A1D5P0F4, R4GHP9, A0A3Q2UD12,A0A3Q2UMJ2, A0A3Q2U4U7, F1NX22, A0A1D5P8I8, A0A1L1RPW4, P13944, P15989,F1P2F0, A0A1D5PGD5, and A0A3Q3AR07.

TABLE 1 Full-length collagen amino acid sequences CollagenAmino Acid Sequence Gallus gallus MAQLLRLFQTLLILLLRDYISAEDGETRAS(chicken) CRTAPADLVFILDGSYSVGPENFEIIKSWL type 21VNITRNEDIGPKFIQVGVVQYSDYPVLEIP alpha 1 LGTHESTENLIKEMESIHYLGGNIKTGRAIcollagen QFAYDHLFAKSSRFLTKIAVVLIDGKSQDE VKDVAAEARKNKITLFAIGVGSEIEEDELKAIANKPSSTYVEYVEDYIAISRIKEVIKQK LCEESVCPTRIPVAARDEKGEDILVGLGVKKRVKKRIQIPTTNAKAYEVISRVDLSELTR NVEPEGLPPSYVEVSTQRFKVKKTWDLWRVLSLDKRPQIAVTINGEEKTLSETTTSLING TQVITFAAPRVKTLFDEGWHQIRLLVTEDFVTLYIDDQEIETKPLHPVLGIYISGLTQIG KYSGKEETVQFDIQKLRIYCDPEQNNRETVCEIPGENGECMNGPSDVGSTPAPCICPPGK QGPPGPKGDPGQPGNHGYPGQPGPDGKPGYQGSAGTPGIPGTPGVQGPRGLPGIKGEPGK DGTKGDRGLPGFPGLHGMPAPKGERGPKGDQGVPGIYGKKGSKGEKGDTGFPGMPGRSGD PGRSGKDGLPGSPGFKGEVGQPGSPGLEGHRGEPGIPGIPGNQGAKGQKGEIGPPGLPGA KGSPGETGLMGPEGSFGLPGAPGPKGDKGEPGLQGKPGSSGAKGEPGGPGAPGEPGYPGI PGTQGIKGDKGSQGESGIQGRKGEKGRQGNPGLQGTEGLRGEQGEKGEKGDPGIRGINGQ KGESGIQGLVGPPGVRGQPGDRGPPGPPGSDGKPAREFSEEFIRQVCSDVLRTQLPVILQ SGRLQNCNHCQSQSASPGLPGPPGPRGPEGPRGFPGLPGNDGVPGLTGIPGRPGARGTRG LPGKNGAKGNQGIGVPGIQGPPGPPGPEGPPGMSKEGRPGERGQPGKDGDRGSPGMPGPV GPPGICDPSLCFSVIVGRDPFRKGPNY(SEQ ID NO: 31) Acipenser MFSFVDSRTVLLLAATQLCLLAVVKCQDVE schrenckiiVQQPGRKGQKGEPGDITDVVGPRGPGGPMG (Japanese PPGEQGPRGERGDKGDKGGPGPRGRDGEPGsturgeon) TPGNPGPPGPPGPNGPPGLGGNFAAQMAGG type 2FDEKAGGAQMGVMQGPMGPMGPRGPPGPTG alpha 1 APGPQGFQGNPGEPGEPGAAGPLGPRGPPGcollagen PSGKPGEDGEAGKPGKSGERGSPGPQGARG FPGTPGLPGIKGHRGYPGLDGAKGEAGAAGSKGEAGSSGENGAPGPMGPRGLPGERGRNG PSGAAGARGNDGLPGPAGPPGPVGPAGAPGFPGSPGSKGEAGPTGARGPEGAQGPRGESG TPGSPGPSGASGNPGTDGIPGAKGSAGAPGIAGAPGFPGPRGPPGPQGATGPLGPKGQQG DPGIPGFKGEHGPKGEHGPAGPQGAPGPAGEEGKRGARGEPGAAGPLGPPGERGAPGNRG FPGQDGLAGPKGAPGERGQPGVGGPKGANGDPGRPGEPGLPGARGLTGRPGDAGPQGKGG PSGAAGEDGRPGPPGPQGARGQPGVMGFPGPKGANGEPGKAGEKGLVGPPGLRGLSGKDG ETGAAGPPGPSGPAGERGEQGPPGPSGFQGLPGPPGPPGEGGKPGDQGVPGEAGAAGRAG PRGERGFPGERGSPGAQGLQGPRGLPGTPGTDGPKGATGPSGALGAQGPPGLQGMPGERG ASGIAGAKGDRGDVGEKGPEGASGKDGSRGLTGPIGPPGPAGPNGEKGESGPSGPPGAAG TRGAPGDRGENGPPGPAGFAGPPGADGQPGAKGEQGEGGQKGDAGAPGPQGPSGAPGPQG PTGVSGPKGARGAQGPPGATGFPGAAGRVGPPGPNGNPGPSGPAGSAGKDGPKGVRGDAG PPGRAGDAGLQGAAGPPGEKGEPGEDGPPGPDGPSGPQGLGGNRGIVGLPGQRGERGFPG LPGPSGEPGKQGAPGGAGDRGPPGPVGPPGLSGPSGEPGREGNPGSDGPPGRDGSAGIKG DRGQTGPAGAPGAPGAPGSPGPVGPTGKQGDRGESGAQGPAGPSGPAGARGMAGPQGPRG DKGEAGETGERGQKGHRGFTGLQGLPGPPGTAGDQGAAGPAGPTGARGPPGPVGPHGKDG SNGQPGPIGPPGPRGRSGEVGPAGPPGNAGPPGPPGPPGPGIDMSAFAGLAAPEKAPDPM RYMRADEASSSLRQHDAEVDATLKSINNQIENIRSPEGSKKNPARTCRDLKLCHPDWKSG DYWIDPNQGCAVDAIKVFCNMESGETCVYPNPASIPRKNWWTSKSADCKHVWFGETMNGG FHFSYGDDSLAPNTASIQMTFLRLLSTEASQNLTYHCKNSIAYMDQSAGNLKKAVLLQGS NDVEIRAEGNSRFTYNVLEDGCTKHTDRWGKTVIEYKSQKTSRLPIVDIAPLDIGGSDQE FGVDIGPVCY (SEQ ID NO: 32)

In some cases, a non-naturally occurring polypeptide (e.g., truncatedcollagen) as described herein may comprise the amino acid sequence ofSEQ ID NO: 31 (or an amino acid sequence having at least 80% (e.g., atleast 85%, at least 90%, at least 95%, at least 98%) sequence identitythereto) with an N-terminal truncation at any amino acid position (e.g.,relative to SEQ ID NO: 31) from amino acid positions 1 to 537; fromamino acid positions 1 to 542; from amino acid positions 1 to 547; fromamino acid positions 1 to 552; from amino acid positions 1 to 557; fromamino acid positions 1 to 562; from amino acid positions 1 to 567; fromamino acid positions 1 to 572; or from amino acid positions 1 to 577. Insome cases, a non-naturally occurring polypeptide (e.g., truncatedcollagen) as described herein may comprise the amino acid sequence ofSEQ ID NO: 31 (or an amino acid sequence having at least 80% (e.g., atleast 85%, at least 90%, at least 95%, at least 98%) sequence identitythereto) with a C-terminal truncation at any amino acid position(relative to SEQ ID NO: 31) from amino acid positions 726 to 957; fromamino acid positions 731 to 957; from amino acid positions 736 to 957;from amino acid positions 741 to 957; from amino acid positions 746 to957; from amino acid positions 751 to 957; from amino acid positions 756to 957; from amino acid positions 761 to 957; from amino acid positions766 to 957; from amino acid positions 769 to 957; from amino acidpositions 774 to 957; from amino acid positions 779 to 957; or fromamino acid positions 784 to 957. In some cases, a non-naturallyoccurring polypeptide as described herein (e.g., a truncated collagen)may comprise both an N-terminal truncation and a C-terminal truncation.For example, a non-naturally occurring polypeptide (e.g., truncatedcollagen) as described herein may comprise the amino acid sequence ofSEQ ID NO: 31 (or an amino acid sequence having at least 80% (e.g., atleast 85%, at least 90%, at least 95%, at least 98%) sequence identitythereto) with an N-terminal truncation at any amino acid position (e.g.,relative to SEQ ID NO: 31) from amino acid positions 1 to 537; fromamino acid positions 1 to 542; from amino acid positions 1 to 547; fromamino acid positions 1 to 552; from amino acid positions 1 to 557; fromamino acid positions 1 to 562; from amino acid positions 1 to 567; fromamino acid positions 1 to 572; or from amino acid positions 1 to 577;and with a C-terminal truncation at any amino acid position (relative toSEQ ID NO: 31) from amino acid positions 726 to 957; from amino acidpositions 731 to 957; from amino acid positions 736 to 957; from aminoacid positions 741 to 957; from amino acid positions 746 to 957; fromamino acid positions 751 to 957; from amino acid positions 756 to 957;from amino acid positions 761 to 957; from amino acid positions 766 to957; from amino acid positions 769 to 957; from amino acid positions 774to 957; from amino acid positions 779 to 957; or from amino acidpositions 784 to 957. In a specific embodiment, a non-naturallyoccurring polypeptide (e.g., truncated collagen) disclosed herein maycomprise the amino acid sequence of SEQ ID NO: 31 (or an amino acidsequence having at least 80% (e.g., at least 85%, at least 90%, at least95%, at least 98%) sequence identity thereto) with an N-terminaltruncation at amino acid position 557 (relative to SEQ ID NO: 31); andwith a C-terminal truncation at amino acid position 746 (relative to SEQID NO: 31). In another specific embodiment, a non-naturally occurringpolypeptide (e.g., truncated collagen) disclosed herein may comprise theamino acid sequence of SEQ ID NO: 31 (or an amino acid sequence havingat least 80% (e.g., at least 85%, at least 90%, at least 95%, at least98%) sequence identity thereto) with an N-terminal truncation at aminoacid position 557 (relative to SEQ ID NO: 31); and with a C-terminaltruncation at amino acid position 769 (relative to SEQ ID NO: 31).

In some cases, a non-naturally occurring polypeptide (e.g., truncatedcollagen) as described herein may comprise the amino acid sequence ofSEQ ID NO: 32 (or an amino acid sequence having at least 80% (e.g., atleast 85%, at least 90%, at least 95%, at least 98%) sequence identitythereto) with an N-terminal truncation at any amino acid position (e.g.,relative to SEQ ID NO: 32) from amino acid positions 1 to 660; fromamino acid positions 1 to 665; from amino acid positions 1 to 670; fromamino acid positions 1 to 675; from amino acid positions 1 to 680; fromamino acid positions 1 to 685; from amino acid positions 1 to 690; fromamino acid positions 1 to 695; or from amino acid positions 1 to 700. Insome cases, a non-naturally occurring polypeptide (e.g., truncatedcollagen) as described herein may comprise the amino acid sequence ofSEQ ID NO: 32 (or an amino acid sequence having at least 80% (e.g., atleast 85%, at least 90%, at least 95%, at least 98%) sequence identitythereto) with a C-terminal truncation at any amino acid position(relative to SEQ ID NO: 32) from amino acid positions 855 to 1420; fromamino acid positions 860 to 1420; from amino acid positions 865 to 1420;from amino acid positions 870 to 1420; from amino acid positions 875 to1420; from amino acid positions 880 to 1420; from amino acid positions885 to 1420; from amino acid positions 890 to 1420; from amino acidpositions 895 to 1420; or from amino acid positions 900 to 1420. In somecases, a non-naturally occurring polypeptide as described herein (e.g.,a truncated collagen) may comprise both an N-terminal truncation and aC-terminal truncation. For example, a non-naturally occurringpolypeptide (e.g., truncated collagen) as described herein may comprisethe amino acid sequence of SEQ ID NO: 32 (or an amino acid sequencehaving at least 80% (e.g., at least 85%, at least 90%, at least 95%, atleast 98%) sequence identity thereto) with an N-terminal truncation atany amino acid position (e.g., relative to SEQ ID NO: 32) from aminoacid positions 1 to 660; from amino acid positions 1 to 665; from aminoacid positions 1 to 670; from amino acid positions 1 to 675; from aminoacid positions 1 to 680; from amino acid positions 1 to 685; from aminoacid positions 1 to 690; from amino acid positions 1 to 695; or fromamino acid positions 1 to 700; and with a C-terminal truncation at anyamino acid position (relative to SEQ ID NO: 32) from amino acidpositions 855 to 1420; from amino acid positions 860 to 1420; from aminoacid positions 865 to 1420; from amino acid positions 870 to 1420; fromamino acid positions 875 to 1420; from amino acid positions 880 to 1420;from amino acid positions 885 to 1420; from amino acid positions 890 to1420; from amino acid positions 895 to 1420; or from amino acidpositions 900 to 1420. In a specific embodiment, a non-naturallyoccurring polypeptide (e.g., truncated collagen) disclosed herein maycomprise the amino acid sequence of SEQ ID NO: 32 (or an amino acidsequence having at least 80% (e.g., at least 85%, at least 90%, at least95%, at least 98%) sequence identity thereto) with an N-terminaltruncation at amino acid position 680 (relative to SEQ ID NO: 32); andwith a C-terminal truncation at amino acid position 880 (relative to SEQID NO: 32).

In some cases, a non-naturally occurring polypeptide (e.g., truncatedcollagen) may comprise any amino acid sequence provided herein. In somecases, a non-naturally occurring polypeptide (e.g., truncated collagen)may consist of any amino acid sequence provided herein. In some cases, anon-naturally occurring polypeptide (e.g., truncated collagen) mayconsist essentially of any amino acid sequence provided herein. Inspecific embodiments, the non-naturally occurring polypeptide has orcomprises an amino acid sequence of any one of SEQ ID NO: 2, SEQ ID NO:4, SEQ ID NO: 6, and SEQ ID NO: 8. In some embodiments, a non-naturallyoccurring polypeptide (e.g., truncated collagen) comprises an amino acidsequence having at least 85%, at least 90%, at least 95%, or at least98% sequence identity to any one of SEQ ID NO: 2, SEQ ID NO: 4, SEQ IDNO: 6, and SEQ ID NO: 8. In some embodiments, the non-naturallyoccurring polypeptide consists of or consists essentially of an aminoacid sequence of any one of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6,and SEQ ID NO: 8.

In some aspects, the non-naturally occurring polypeptide may include anychimeric collagen that includes at least one non-continuous collagenfragment. For example, the non-naturally occurring polypeptide can be achimeric collagen in which a portion of N-terminus collagen iscontiguous with a portion of C-terminus collagen where the portion ofN-terminus collagen and the portion of C-terminus collagen are notcontiguous in the natural or naturally-present corresponding collagens.In another example, the non-naturally occurring polypeptide can be achimeric collagen in which a portion of C-terminus collagen iscontiguous with a portion of N-terminus collagen (e.g., in a flipped orreverse order—C terminus collagen is located in the N-terminus of theportion of N-terminus collagen) where the portion of C-terminus collagenand the portion of N-terminus collagen are contiguous or non-contiguousin the natural or naturally-present corresponding collagens. In anotherexample, the non-naturally occurring polypeptide can be a chimericcollagen in which one portion of a collagen polypeptide is contiguouswith a portion of a second collagen polypeptide (e.g., a collagenmolecule comprising a portion of a collagen from a first speciescontiguous with a portion of a collagen from a second species is achimeric collagen, etc.).

Exemplary amino acid sequences of or nucleic acid sequences encoding therecombinant proteins are provided below:

A nucleotide sequence encoding a truncated collagen type 21 alpha 1polypeptide from Gallus gallus (chicken) SEQ ID NO: 1GATACCGGTTTTCCGGGTATGCCTGGTCGTAGCGG TGATCCGGGTCGTAGCGGTAAAGATGGTCTGCCTGGTAGCCCGGGTTTTAAAGGTGAAGTTGGTCAGCCA GGTAGCCCTGGTCTGGAAGGTCATCGTGGTGAACCGGGTATTCCAGGTATTCCGGGTAATCAGGGTGCAA AAGGTCAGAAAGGCGAAATTGGTCCTCCGGGTCTGCCAGGTGCCAAAGGTTCTCCGGGTGAAACCGGTCT GATGGGTCCTGAAGGTAGCTTTGGCCTGCCTGGTGCACCGGGTCCGAAAGGTGACAAAGGTGAACCTGGT CTGCAGGGTAAACCGGGTAGCAGCGGTGCAAAAGGCGAACCAGGTGGTCCGGGTGCTCCGGGTGAACCAG GCTATCCGGGTATTCCTGGTACTCAGGGTATTAAAGGCGATAAAGGTAGCCAGGGTGAAAGCGGTATTCA GGGTCGTAAGGGTGAAAAAGGCCGTCAGGGTAATCCAGGCCTGCAGGGCACCGAAGGTCTGCGTGGCGAA CAGGGCGAAAAAGGTGAGAAGGGTGACCCAGGCATTCGT Amino acid sequence of a truncatedcollagen type 21 alpha 1 polypeptide from Gallus gallus (chicken)SEQ ID NO: 2 DTGFPGMPGRSGDPGRSGKDGLPGSPGFKGEVGQPGSPGLEGHRGEPGIPGIPGNQGAKGQKGEIGPPGL PGAKGSPGETGLMGPEGSFGLPGAPGPKGDKGEPGLQGKPGSSGAKGEPGGPGAPGEPGYPGIPGTQGIK GDKGSQGESGIQGRKGEKGRQGNPGLQGTEGLRGEQGEKGEKGDPGIR A nucleotide sequence encoding atruncated collagen type 21 alpha 1 polypeptide from Gallus gallus(chicken) SEQ ID NO: 3 GATACTGGTTTCCCGGGGATGCCTGGGCGCTCAGGTGATCCGGGGCGTAGTGGAAAAGACGGTCTGCCGG GGTCCCCGGGCTTTAAGGGTGAGGTGGGTCAGCCCGGTAGTCCAGGTTTAGAAGGTCACCGCGGAGAGCC CGGGATTCCAGGCATTCCTGGCAACCAGGGTGCCAAGGGACAGAAAGGCGAAATTGGTCCGCCCGGCCTA CCGGGCGCGAAAGGTTCTCCTGGTGAAACCGGTCTCATGGGTCCGGAAGGTAGCTTCGGCCTGCCCGGCG CACCTGGTCCGAAGGGCGATAAGGGGGAGCCTGGGCTGCAAGGTAAACCGGGTAGTTCTGGCGCCAAAGG TGAACCCGGCGGTCCCGGTGCGCCAGGGGAACCAGGTTATCCTGGTATTCCTGGAACCCAAGGAATTAAA GGTGACAAAGGCTCACAGGGCGAAAGTGGTATACAGGGTCGCAAGGGCGAAAAAGGACGTCAGGGCAATC CAGGCCTGCAGGGTACTGAAGGCCTGCGTGGAGAACAGGGTGAGAAAGGTGAAAAAGGAGATCCTGGTAT TCGC Amino acid sequence ofa truncated collagen type 21 alpha 1 polypeptide from Gallus gallus(chicken) SEQ ID NO: 4 DTGFPGMPGRSGDPGRSGKDGLPGSPGFKGEVGQPGSPGLEGHRGEPGIPGIPGNQGAKGQKGEIGPPGL PGAKGSPGETGLMGPEGSFGLPGAPGPKGDKGEPGLQGKPGSSGAKGEPGGPGAPGEPGYPGIPGTQGIK GDKGSQGESGIQGRKGEKGRQGNPGLQGTEGLRGEQGEKGEKGDPGIR The nucleotide sequence encoding atruncated collagen type 21 alpha 1 polypeptide from Gallus gallus(chicken) SEQ ID NO: 5 GATACTGGTTTCCCGGGGATGCCTGGGCGCTCAGGTGATCCGGGGCGTAGTGGAAAAGACGGTCTGCCGG GGTCCCCGGGCTTTAAGGGTGAGGTGGGTCAGCCCGGTAGTCCAGGTTTAGAAGGTCACCGCGGAGAGCC CGGGATTCCAGGCATTCCTGGCAACCAGGGTGCCAAGGGACAGAAAGGCGAAATTGGTCCGCCCGGCCTA CCGGGCGCGAAAGGTTCTCCTGGTGAAACCGGTCTCATGGGTCCGGAAGGTAGCTTCGGCCTGCCCGGCG CACCTGGTCCGAAGGGCGATAAGGGGGAGCCTGGGCTGCAAGGTAAACCGGGTAGTTCTGGCGCCAAAGG TGAACCCGGCGGTCCCGGTGCGCCAGGGGAACCAGGTTATCCTGGTATTCCTGGAACCCAAGGAATTAAA GGTGACAAAGGCTCACAGGGCGAAAGTGGTATACAGGGTCGCAAGGGCGAAAAAGGACGTCAGGGCAATC CAGGCCTGCAGGGTACTGAAGGCCTGCGTGGAGAACAGGGTGAGAAAGGTGAAAAAGGAGATCCTGGTAT TCGCGGCATTAACGGTCAAAAGGGTGAAAGTGGGATACAAGGTCTTGTCGGTCCGCCCGGAGTTAGAGGC CAGAmino acid sequence of a truncated collagen type 21 alpha 1 polypeptidefrom Gallus gallus (chicken) SEQ ID NO: 6DTGFPGMPGRSGDPGRSGKDGLPGSPGFKGEVGQP GSPGLEGHRGEPGIPGIPGNQGAKGQKGEIGPPGLPGAKGSPGETGLMGPEGSFGLPGAPGPKGDKGEPG LQGKPGSSGAKGEPGGPGAPGEPGYPGIPGTQGIKGDKGSQGESGIQGRKGEKGRQGNPGLQGTEGLRGE QGEKGEKGDPGIRGINGQKGESGIQGLVGPPGVRGQ The nucleotide sequence encoding a truncated collagen type 2 alpha 1polypeptide from Acipenser schrenckii (Japanese sturgeon) SEQ ID NO: 7GTCTGCAGGGTATGCCTGGTGAACGTGGTGCAAGC GGTATTGCCGGTGCAAAAGGTGATCGTGGTGATGTTGGTGAAAAAGGTCCGGAAGGTGCCAGCGGTAAAG ATGGTAGCCGTGGTCTGACCGGTCCGATTGGTCCGCCTGGTCCGGCAGGTCCGAATGGCGAAAAAGGTGA AAGCGGTCCGAGCGGTCCTCCGGGTGCAGCAGGTACTCGTGGTGCACCGGGTGATCGCGGTGAAAATGGT CCACCGGGTCCTGCCGGTTTTGCAGGTCCGCCAGGTGCAGATGGTCAGCCTGGTGCCAAAGGCGAACAAG GCGAAGGTGGTCAGAAAGGTGATGCAGGCGCTCCGGGTCCGCAGGGTCCTTCTGGTGCACCTGGTCCTCA GGGTCCGACCGGTGTTTCTGGTCCGAAAGGCGCACGTGGTGCCCAGGGTCCACCTGGTGCGACCGGTTTT CCTGGCGCAGCAGGTCGTGTTGGTCCTCCAGGTCCTAATGGTAATCCGGGTCCAAGCGGTCCTGCAGGTA GCGCAGGCAAAGATGGTCCTAAAGGTGTACGCGGTGATGCTGGTCCTCCTGGCCGTGCCGGTGATGCCGG T Amino acid sequence of a truncatedcollagen type 2 alpha 1 polypeptide from Acipenser schrenckii(Japanese sturgeon) SEQ ID NO: 8 GLQGMPGERGASGIAGAKGDRGDVGEKGPEGASGKDGSRGLTGPIGPPGPAGPNGEKGESGPSGPPGAAG TRGAPGDRGENGPPGPAGFAGPPGADGQPGAKGEQGEGGQKGDAGAPGPQGPSGAPGPQGPTGVSGPKGA RGAQGPPGATGFPGAAGRVGPPGPNGNPGPSGPAGSAGKDGPKGVRGDAGPPGRAGDAG The nucleotide sequence encoding asecretion signal sequence named Secretion Signal Sequence 1 SEQ ID NO: 9ATGAAAAAGATTTGGCTGGCGCTGGCTGGTTTAGT TTTAGCGTTTAGCGCATCGGCGAmino acid sequence of a Secretion Signal Sequence 1 SEQ ID NO: 10MKKIWLALAGLVLAFSASA The nucleotide sequence encodinga secretion signal sequence named Secretion Signal Sequence 2SEQ ID NO: 11 ATGAAAAAAGGTTTCATGCTGTTCACCCTCCTCGCTGCGTTCTCTGGTTTCGCGCAGGCT Amino acid sequence of a SecretionSignal Sequence 2 SEQ ID NO: 12 MKKGFMLFTLLAAFSGFAQAThe nucleotide sequence encoding a secretion signal sequence namedSecretion Signal Sequence 3 SEQ ID NO: 13ATGATGATCACCCTGCGTAAACTGCCGCTGGCTGT TGCTGTTGCTGCTGGTGTTATGTCTGCTCAGGCTATGGCT Amino acid sequence of a Secretion Signal Sequence 3 SEQ ID NO: 14MMITLRKLPLAVAVAAGVMSAQAMA The nucleotide sequence encoding asecretion signal sequence named Secretion Signal Sequence 4SEQ ID NO: 15 ATGAAAAAAACCGCTATCGCTATCGCTGTTGCTCTGGCTGGTTTCGCTACCGTTGCTCAGGCT Amino acid sequence of a SecretionSignal Sequence 4 SEQ ID NO: 16 MKKTAIAIAVALAGFATVAQAThe nucleotide sequence encoding a secretion signal sequence namedSecretion Signal Sequence 5 SEQ ID NO: 17ATGAAAGTTAAAGTTCTGTCTCTGCTGGTTCCGGC TCTGCTGGTTGCTGGTGCTGCTAACGCTAmino acid sequence of a Secretion Signal Sequence 5 SEQ ID NO: 18MKVKVLSLLVPALLVAGAANA The nucleotide sequence encoding asecretion signal sequence named Secretion Signal Sequence 6SEQ ID NO: 19 ATGAAACATCCTGTCTCTGTCTATGGTTGCTCTGTCTCTGTCTCTGGCTCTGGGTTCTGTTTCTGTTACC GCTAmino acid sequence of a Secretion Signal Sequence 6 SEQ ID NO: 20MKKNILSLSMVALSLSLALGSVSVTA The nucleotide sequence encoding asecretion signal sequence named Secretion Signal Sequence 7SEQ ID NO: 21 ATGCTGAACCCGAAAGTTGCTTACATGGTTTGGATGACCTGCCTGGGTCTGACCCTGCCGTCTCAGGCT Amino acid sequence of a SecretionSignal Sequence 7 SEQ ID NO: 22 MLNPKVAYMVWMTCLGLTLPSQAThe nucleotide sequence encoding a secretion signal sequence namedSecretion Signal Sequence 8 SEQ ID NO: 23ATGAAACAGGCTCTGCGTGTAGCGTTCGGTTTCCT GATACTGTGGGCTTCTGTTCTGCACGCTAmino acid sequence of a Secretion Signal Sequence 8 SEQ ID NO: 24MKQALRVAFGFLILWASVLHA A codon-optimized nucleotide sequenceencoding a truncated collagen type 2 alpha 1 polypeptide fromAcipenser schrenckii (Japanese sturgeon) SEQ ID NO: 25GGTCTGCAGGGTATGCCGGGTGAACGTGGTGCCAG CGGTATTGCAGGTGCCAAAGGTGATCGTGGTGATGTTGGTGAAAAAGGTCCGGAAGGTGCAAGCGGTAAA GATGGTAGCCGTGGTCTGACCGGTCCGATTGGTCCGCCGGGTCCGGCCGGTCCGAATGGTGAAAAAGGTG AAAGCGGTCCGAGCGGTCCGCCGGGTGCAGCCGGTACCCGTGGTGCACCGGGTGATCGTGGTGAAAATGG TCCGCCGGGTCCGGCCGGTTTTGCAGGTCCGCCGGGTGCCGATGGTCAGCCGGGTGCAAAAGGTGAACAG GGTGAAGGTGGTCAGAAAGGTGATGCCGGTGCACCGGGTCCGCAGGGTCCGAGCGGTGCCCCGGGTCCGC AGGGTCCGACCGGTGTTAGCGGTCCGAAAGGTGCACGTGGTGCCCAGGGTCCGCCGGGTGCAACCGGTTT TCCGGGTGCCGCAGGTCGTGTTGGTCCGCCGGGTCCGAATGGTAATCCGGGTCCGAGCGGTCCGGCAGGT AGCGCCGGTAAAGATGGTCCGAAAGGTGTTCGTGGTGATGCAGGTCCGCCGGGTCGTGCCGGTGATGCAG GTTAAA codon-optimized nucleotide sequence encoding a truncated collagen type2 alpha 1 polypeptide from Acipenser schrenckii (Japanese sturgeon)SEQ ID NO: 26 GGCCTGCAAGGCATGCCAGGCGAGCGCGGCGCGTCTGGCATCGCGGGCGCGAAGGGCGACCGCGGCGACG TGGGCGAGAAGGGCCCTGAGGGCGCGTCCGGCAAGGACGGCTCTCGCGGCCTGACAGGCCCAATCGGCCC TCCAGGCCCTGCGGGCCCAAACGGCGAGAAGGGCGAGTCCGGCCCTTCTGGCCCACCTGGCGCGGCGGGC ACACGCGGCGCGCCAGGCGACCGCGGCGAGAACGGCCCTCCAGGCCCTGCGGGCTTCGCGGGCCCACCTG GCGCGGACGGCCAACCAGGCGCGAAGGGCGAGCAAGGCGAGGGCGGCCAAAAGGGCGACGCGGGCGCGCC TGGCCCACAAGGCCCTTCTGGCGCGCCAGGCCCTCAAGGCCCAACAGGCGTGTCCGGCCCTAAGGGCGCG CGCGGCGCGCAAGGCCCACCTGGCGCGACAGGCTTCCCAGGCGCGGCGGGCCGCGTGGGCCCTCCAGGCC CTAACGGCAACCCAGGCCCTTCTGGCCCAGCGGGCTCCGCGGGCAAGGACGGCCCTAAGGGCGTGCGCGG CGACGCGGGCCCACCTGGCCGCGCGGGCGACGCGGGCTGA A codon-optimized nucleotide sequenceencoding a truncated collagen type 2 alpha 1 polypeptide fromAcipenser schrenckii (Japanese sturgeon) SEQ ID NO: 27GGTTTGCAAGGTATGCCAGGGGAACGGGGTGCGTC CGGGATAGCCGGGGCAAAAGGTGATCGAGGCGATGTAGGAGAAAAAGGCCCAGAAGGGGCGTCAGGTAAG GACGGATCTCGCGGCTTGACGGGACCTATCGGGCCTCCAGGTCCCGCCGGCCCTAATGGGGAAAAAGGCG AGAGTGGGCCGTCTGGTCCGCCCGGCGCCGCTGGCACACGTGGAGCGCCGGGCGATCGTGGTGAGAACGG ACCACCGGGTCCTGCTGGTTTTGCGGGACCTCCGGGAGCAGACGGCCAGCCGGGCGCTAAAGGTGAACAG GGTGAAGGTGGCCAAAAAGGCGATGCAGGCGCACCGGGTCCGCAGGGCCCTTCAGGTGCACCGGGTCCAC AGGGCCCAACTGGCGTTTCAGGGCCGAAAGGCGCAAGAGGTGCTCAGGGTCCGCCCGGGGCAACTGGGTT TCCTGGAGCGGCCGGCCGTGTTGGACCTCCGGGGCCGAACGGAAACCCTGGACCGTCTGGACCAGCCGGT TCAGCGGGTAAGGATGGTCCTAAGGGTGTAAGGGGTGACGCAGGTCCCCCTGGACGTGCAGGGGATGCGG GGTAGA codon-optimized nucleotide sequence encoding a truncated collagen type2 alpha 1 polypeptide from Acipenser schrenckii (Japanese sturgeon)SEQ ID NO: 28 GGGTTACAAGGTATGCCGGGAGAACGTGGAGCGTCAGGAATTGCTGGGGCCAAAGGTGATCGTGGTGATG TTGGCGAGAAAGGGCCCGAAGGCGCATCTGGTAAAGATGGCTCACGCGGGTTAACTGGACCAATCGGACC ACCAGGCCCCGCTGGGCCTAATGGTGAAAAGGGTGAAAGTGGCCCTTCTGGACCCCCAGGAGCCGCCGGT ACACGTGGAGCGCCAGGCGATCGTGGCGAAAACGGACCGCCCGGACCTGCAGGTTTTGCGGGACCCCCTG GAGCAGACGGCCAACCAGGAGCAAAAGGTGAGCAAGGTGAAGGTGGACAAAAGGGAGATGCCGGAGCGCC AGGCCCCCAAGGCCCATCAGGAGCTCCAGGACCTCAAGGTCCAACTGGTGTATCAGGGCCTAAGGGTGCG CGCGGCGCTCAAGGACCGCCTGGCGCAACTGGCTTTCCGGGAGCTGCTGGTCGTGTGGGCCCGCCTGGCC CAAACGGAAATCCAGGCCCTTCAGGCCCGGCGGGCTCAGCCGGAAAAGACGGTCCGAAGGGAGTCCGTGG AGATGCGGGACCGCCAGGACGCGCTGGCGATGCAGGCTAA A codon-optimized nucleotide sequenceencoding a truncated collagen type 2 alpha 1 polypeptide fromAcipenser schrenckii (Japanese sturgeon) SEQ ID NO: 29GGTTTACAGGGAATGCCAGGGGAACGCGGCGCCTC AGGGATTGCCGGTGCTAAAGGAGATCGTGGCGACGTGGGTGAAAAGGGTCCCGAGGGAGCATCAGGTAAG GATGGTTCCCGTGGTTTGACGGGACCTATTGGACCTCCGGGTCCTGCAGGTCCGAACGGCGAAAAGGGGG AAAGCGGGCCTAGTGGTCCACCCGGCGCCGCAGGTACCCGTGGTGCCCCAGGCGACCGCGGGGAGAATGG ACCGCCTGGCCCTGCCGGTTTTGCGGGTCCTCCAGGAGCCGATGGGCAGCCCGGTGCAAAAGGAGAGCAG GGAGAGGGAGGTCAAAAGGGAGATGCCGGCGCCCCGGGCCCTCAGGGACCAAGCGGTGCGCCAGGCCCCC AGGGTCCTACGGGTGTTAGCGGGCCGAAAGGCGCACGCGGAGCGCAGGGCCCACCTGGTGCAACAGGCTT CCCAGGAGCTGCGGGGCGCGTCGGACCTCCGGGACCCAATGGAAACCCAGGTCCGTCAGGGCCGGCAGGC TCCGCAGGGAAAGATGGTCCCAAAGGCGTGCGTGGAGACGCAGGGCCCCCCGGACGCGCCGGCGATGCGG GATAAA codon-optimized nucleotide sequence encoding a truncated collagen type21 polypeptide from Gallus gallus SEQ ID NO: 30GATACTGGTTTCCCGGGGATGCCTGGGCGCTCAGG TGATCCGGGGCGTAGTGGAAAAGACGGTCTGCCGGGGTCCCCGGGCTTTAAGGGTGAGGTGGGTCAGCCC GGTAGTCCAGGTTTAGAAGGTCACCGCGGAGAGCCCGGGATTCCAGGCATTCCTGGCAACCAGGGTGCCA AGGGACAGAAAGGCGAAATTGGTCCGCCCGGCCTACCGGGCGCGAAAGGTTCTCCTGGTGAAACCGGTCT CATGGGTCCGGAAGGTAGCTTCGGCCTGCCCGGCGCACCTGGTCCGAAGGGCGATAAGGGGGAGCCTGGG CTGCAAGGTAAACCGGGTAGTTCTGGCGCCAAAGGTGAACCCGGCGGTCCCGGTGCGCCAGGGGAACCAG GTTATCCTGGTATTCCTGGAACCCAAGGAATTAAAGGTGACAAAGGCTCACAGGGCGAAAGTGGTATACA GGGTCGCAAGGGCGAAAAAGGACGTCAGGGCAATCCAGGCCTGCAGGGTACTGAAGGCCTGCGTGGAGAA CAGGGTGAGAAAGGTGAAAAAGGAGATCCTGGTATTCGC

In some embodiments, the non-naturally occurring polypeptide comprisesan amino acid sequence of any one of SEQ ID NOs: 2, 4, 6, and 8. In someembodiments, the non-naturally occurring polypeptide comprises an aminoacid sequence having a sequence identity of at least 80%, at least 85%,at least 90%, at least 95%, or at least 98% thereof, or the like, to theamino acid sequence of any one of SEQ ID NOs: 2, 4, 6, and 8.Alternatively and/or additionally, the non-naturally occurringpolypeptide is encoded by a nucleic acid sequence of any one of SEQ IDNOs: 1, 3, 5, 7, and 25-30. In some embodiments, the non-naturallyoccurring polypeptide is encoded by a nucleic acid having sequenceidentity of at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, or at least 98% thereof, or thelike, to the nucleic acid sequence of any one of SEQ ID NOs: 1, 3, 5, 7,and 25-30.

In some aspects, the non-naturally occurring polypeptides providedherein may or may not contain one or more domains from natural collagen.FIG. 6 depicts an alignment of exemplary non-naturally occurringpolypeptides (e.g., truncated collagens) of the disclosure with thecorresponding naturally occurring collagen. The top panel depicts analignment of a non-naturally occurring polypeptide of SEQ ID NO: 2 andSEQ ID NO: 6 with Gallus gallus type 21 alpha 1 collagen (e.g., SEQ IDNO: 31). The bottom panel depicts an alignment of a non-naturallyoccurring polypeptide of SEQ ID NO: 8 with Acipenser schrenckii type 2alpha 1 collagen. FIG. 6 demonstrates that non-naturally occurringpolypeptides may have one or more domains found in natural collagen(e.g., collagen triple helix repeat domains). FIG. 6 furtherdemonstrates that non-naturally occurring polypeptides may lack one ormore domains found in natural collagen (e.g., Von Willebrand factor typeA (vWA) domain, laminin G domain, fibrillar collagen C-terminal domain).In some aspects, a non-naturally occurring polypeptide provided hereinmay contain one or more collagen triple helix repeat domains. In someaspects, a non-naturally occurring polypeptide provided herein may lackone or more of a Von Willebrand factor type A (vWA) domain, a laminin Gdomain, and a fibrillar collagen C-terminal domain).

In some embodiments, the non-naturally occurring polypeptide (e.g.,recombinant polypeptide) includes a secretion signal sequence. Anysuitable secretion signal sequence (e.g., hydrophobic signalingpeptides, Sec signal peptides, Tat signal peptides, etc.) that caninduce the non-naturally occurring polypeptide (e.g., recombinantpolypeptide) to be secreted to the periplasmic and/or extracellularspace (e.g., when produced in a recombinant host cell). Exemplarysecretion signal sequences includes a peptide having an amino acidsequence of any one of SEQ ID NOs: 10, 12, 14, 16, 18, 20, 22, and 24.Alternatively and/or additionally, the secretion signal sequenceincludes a peptide encoded by a nucleic acid sequence of any one of SEQID NOs: 9, 11, 13, 15, 17, 19, 21, and 23. The secretion signal sequenceis preferably located in the N-terminus of the non-naturally occurringpolypeptide (e.g., recombinant polypeptide). Yet, it is contemplatedthat the secretion signal sequence can be located at other thanN-terminus where the secretion signal sequence remains functional.

The non-naturally occurring polypeptide (e.g., recombinant polypeptide)as described herein can be expressed or generated via a nucleic acidsequence encoding the non-naturally occurring polypeptide (e.g.,recombinant polypeptide). Thus, another aspect of the disclosureincludes an expression vector comprising a nucleic acid sequenceencoding the non-naturally occurring polypeptide (e.g., recombinantpolypeptide). In some embodiments, the expression vector is a bacterialexpression vector. In some embodiments, the expression vector is a yeastexpression vector. In some embodiments, the expression vector is aninsect expression vector. Any suitable expression vector that can inducethe protein expression from the inserted nucleic acid encoding thenon-naturally occurring polypeptide (e.g., recombinant polypeptide).Exemplary bacterial expression vectors may include pGEX vectors whereglutathione S-transferase is used as a fusion partner and geneexpression is under the control of the tac promoter, or pET vectors(e.g., pET28 vector, etc.) which uses a T7 promoter. Exemplary yeastexpression vectors may include pPIC vectors, which uses the AOX1promoter inducible with methanol. In some embodiments, the expressionvector is in a plasmid form (e.g., including bacterial artificialchromosome form, etc.) that are independently present in the host cell(e.g., cells expressing the recombinant polypeptide). In someembodiments, the expression vector is stably integrated into thechromosome of the host cell via random or targeted integration.

In some embodiments, the nucleic acid sequence encoding thenon-naturally occurring polypeptide (e.g., recombinant polypeptide) iscodon-optimized to be expressed in non-animal cells, preferably inbacterial cells. As used herein, “codon-optimized” means that the codoncomposition is improved for expression in the heterologous cells (e.g.,microbial cells, bacterial cells, etc.) without altering the encodedamino acid sequences. Non-limiting examples of codon-optimized nucleicacid sequences (e.g., encoding a non-naturally occurring polypeptide asdescribed herein) include SEQ ID NOs: 25-30.

In some embodiments, the expression vector may include one or moreselection agent. The selection agents include certain sugars includinggalactose containing sugars or antibiotics including ampicillin,hygromycin, G418 and others. Enzymes that are used to confer resistanceto the selection agent include β-galactosidase or a β-lactamase.Alternatively and/or additionally, the expression vector includes aninducible promoter or a constitutive promoter (e.g., CMV promoter, etc.)such that the nucleic acid encoding the recombinant protein isoperatively linked to the inducible promoter or the constitutivepromoter. For example, the expression vector may includetetracycline-inducible promoter pTET, araC-ParaBAD inducible promoter,or IPTG inducible lac promoter. As used herein, “operatively linked”promoter and nucleic acid means that the expression of the nucleic acid(e.g., transcription, translation, etc.) is at least under partialcontrol of the promoter.

In some embodiments, the nucleic acid encoding the non-naturallyoccurring polypeptide (e.g., recombinant polypeptide) (e.g., a nucleicacid of any one of SEQ ID NOs: 1, 3, 5, 7, and 25-30), and theexpression vector may have an overlap of from 20 to 50 bp long, from 20to 40 bp long, from 20 to 30 bp long, or from 30 to 40 bp long. Suchoverlap can be added using PCR with a DNA polymerase (e.g., PRIMESTAR®GXL polymerase(www.takarabio.com/products/per/gc-rich-per/primestar-gxl-dna-polymerase)).Opened expression vector and the insert nucleic acid encoding thenon-naturally occurring polypeptide (e.g., recombinant polypeptide) canbe assembled together into the final plasmid using any suitable cloningsystem (e.g., IN-FUSION® Cloning(www.takarabio.com/products/cloning/in-fusion-cloning) or SGI Gibsonassembly(us.vwr.com/store/product/17613857/gibson-assembly-hifi-1-step-kit-synthetic-genomics-inc)).

Such prepared expression vector (or plasmid) can be used to generategenetically engineered or modified organisms, or a recombinant cell toproduce the non-naturally occurring polypeptides described herein (e.g.,collagens, truncated collagens, or collagen fragments). Preferably, therecombinant cells contain at least one copy of a plasmid or a stablyintegrated heterologous nucleic acid sequence encoding the non-naturallyoccurring polypeptide (e.g., collagens, truncated collagens, or collagenfragments, preferably collagens, truncated collagens, or collagenfragments of, or derived from, Gallus gallus collagen and/or Acipenserschrenckii collagen). In some embodiments, the recombinant cell is amicrobial cell. For example, where the expression vector is bacterialexpression vector, the expression vector can be inserted into (e.g., viaany suitable transformation method) the bacterial cells for proteinexpression (e.g., Escherichia coli including BL-21 cells, etc.) to beindependently present in the cytoplasm of the bacteria (e.g., as aplasmid form) or to be at least temporarily and/or stably integratedinto the bacterial chromosome.

Consequently, the transformed cells can be cultivated in a suitablemedia. Preferably, the suitable media includes a minimal media and thecells are frozen in 1.5 aliquots with vegetable glycerin at a ratio of50:50 of cells of cells to glycerin. For protein expression, one vial ofthe frozen cultured cells can be cultured in a suitable amount ofbacteria culture media (e.g., minimal media, 50 ml, 100 ml, etc.) for atleast 6 hours, at least 8 hours, at least 10 hours, at least 12 hours,at least overnight at at least 36° C., preferably at about 37° C. bycontinuously shaking the culture (e.g., at least 100 rpm, at least 200rpm, at least 250 rpm, etc.). Table 2 and Table 3 show the exemplaryformulation of the minimal media that can be used for cell cultivationand culture.

TABLE 2 Minimal Media Formulation 1) Autoclave 5 L of 550 g/kg Glucosesyrup at concentration in DI water. (VWR, product #97061-170). 2)Autoclave in 3946 20 g (NH₄)₂HPO₄. (VWR, product mL of DI water and #97061-932). add 66.5 g KH₂PO₄. (VWR, product # 97062-348). 22.5 gH₃C₆H₅O₇. (VWR, product #BDH9228-2.5 KG). 8.85 g MgSO₄•7H₂O. (VWR,product # 97062-134). 10 mL of 1000x Trace metals formulation Afterautoclaving, add: 118 g of (1) to (2) 5 mL of 25 mg/mL Kanamycin Sulfate(VWR-V0408) Use 28% NH₄OH (VWR, product #BDH3022) to adjust pH to 6.1.

TABLE 3 Trace metals formulation Ferrous Sulfate Heptahydrate 27.8 g/L(Spectrum, 7782-63-0) Zinc Sulfate heptahydrate 2.88 g/L (Spectrum,7446-20-0) Calcium chloride dihydrate 2.94 g/L (Spectrum, 2971347)Sodium molybdate dihydrate 0.48 g/L (Spectrum, 10102-40-6) Manganesechloride tetrahydrate 1.26 g/L (Spectrum, 13446-34-9) Sodium selenite0.35 g/L (Spectrum, 10102-18-8) Boric acid 0.12 g/L (Spectrum,10043-35-3)

In some embodiments, transformed cells can then be transferred to alarger volume of growth media (e.g., minimal media) and grown for atleast 4 hours, at least 5 hours, at least 6 hours, at least 7 hours, atleast 8 hours, from 5 to 10 hours, from 5 to 9 hours, from 6 to 9 hours,and/or alternatively until the cell density in the media reaches opticaldensity (OD) of 600.

Additionally, fermentation process can be performed at varioustemperature ranging from 22° C. to 33° C., from 29° C. to 33° C., from30° C. to 32° C., from 23° C. to 29° C., or from 25° C. to 28° C. Insome embodiments, the temperature of the fermentation can be maintainedat a constant temperature and immediately upon completion offermentation the non-naturally occurring polypeptide can be purified.Alternatively, the temperature of the fermentations can be maintainedfor a desired period of time and when cell densities of OD600 of 10-20are reached, then the temperature can be reduced to induce proteinproduction. In such embodiments, typically, the temperature is reducedfrom 28° C. to 25° C. During the fermentation, protein expression in thebacteria can be induced by adding induction reagent. For example, wherethe expression vector contains lac promoter and the nucleic acidencoding the non-naturally occurring polypeptide (e.g., truncatedcollagen, collagen fragments, or collagen) is under the control of thelac promoter, the expression of the nucleic acid can be induced byadding isopropyl β-d-1-thiogalactopyranoside (IPTG) at a concentrationranging from 0.1-1.5 mM, from 0.1-1.0 mM, or from 0.1-0.5 mM.Fermentation can be continued for 20-24 hours, or in some embodiments,for 40-60 hours.

It is contemplated that such generated recombinant cells (e.g.,recombinant bacteria transformed with the expression vector)intracellularly express the non-naturally occurring polypeptides (e.g.,truncated collagen, collagen fragments, or collagen) encoded by thenucleic acids in the expression vector. Such intracellularly expressedpolypeptides (e.g., truncated collagen, collagen fragments, or collagen)can then be secreted (via a secretion signal sequence) to theextracellular space (e.g., into a culture media). Thus, in someembodiments, the culture media can contain secreted recombinant protein(e.g., truncated collagen, collagen fragments, or collagen) encoded bythe nucleic acids.

Thus, another aspect of the disclosure includes a composition includingthe non-naturally occurring polypeptide (e.g., recombinant collagen,truncated collagen, collagen fragments, or collagen) encoded by thenucleic acids. In some embodiments, the composition may include therecombinant cell comprising an integrated heterologous nucleic acidsequence encoding a non-naturally occurring polypeptide (e.g., collagen,a truncated collagen, or fragment thereof), and/or the culture medium(e.g., growth media, cultivation media, etc.) for the recombinant cell.

Alternatively and/or additionally, the composition may include purifiedrecombinant proteins from the recombinant cells and/or the culturemedium. In some embodiments, the recombinant proteins are purified fromthe culture medium where the recombinant cells grow and secrete therecombinant proteins thereto. In some embodiments, the recombinantprotein is coupled with a tag (e.g., histidine tag, etc) such that therecombinant protein can be purified using affinity purification is knownas immobilized metal affinity chromatography (IMAC). Alternatively, therecombinant protein can be purified via column chromatography. Forexample, the recombinant protein can be purified by acid treatment ofhomogenized growth media. In such example, the pH of the growth media(e.g., fermentation broth) can be decreased to from 3 to 3.5 using 5-50%sulfuric Acid. The recombinant cells are then separated usingcentrifugation. Supernatant of the acidified broth can be tested on apolyacrylamide gel and determined whether it contains the recombinantprotein in relatively high abundance compared to starting pellet. Therecombinant protein slurry obtained is generally high in salts. Toobtain volume and salt reduction, concentration and diafiltration stepscan be performed using filtration steps. For example, the filtrationstep can be performed using EMD Millipore Tangential Flow Filtrationsystem with ultrafiltration cassettes of 0.1 m² each. Total area offiltration in this example can be 0.2 m² using two cassettes inparallel. A volume reduction of 5× and a salt reduction of 19× can beachieved in the TFF stage. Final collagen slurry can be run on anSDS-PAGE gel to confirm presence of the recombinant protein. Thepurified recombinant protein can then be analyzed on an SDS-PAGE gel toidentify a corresponding thick and clear band observed at the expectedsizes for each respective protein. Quantification of titers and puritycan be further conducted using reverse phase and size exclusion HPLCchromatography. It is preferred that the purity of the purifiedrecombinant proteins is at least at least 80%, at least 85%, at least90%, at least 95%, or at least 99%.

In some embodiments, the composition including the non-naturallyoccurring polypeptides provided herein (e.g., recombinant proteinsand/or purified recombinant proteins) can be formulated for consumptionby an individual (e.g., a human, a patient, a person, an animal, etc.).In some embodiments, the non-naturally occurring polypeptides (e.g.,recombinant proteins and/or purified recombinant proteins) can beformulated for oral consumption as nutraceutical supplements. In someembodiments, the non-naturally occurring polypeptides (e.g., recombinantproteins and/or purified recombinant proteins) can be formulated fororal consumption as a food product or a food ingredient. In someembodiments, the non-naturally occurring polypeptides can be formulatedas a protein supplement. Optionally, in such embodiments, thenon-naturally occurring polypeptides (e.g., recombinant proteins and/orpurified recombinant proteins) can be mixed with at least one of acarrier molecule, a preservative, and/or additional edible ingredients.Thus, for example, the composition may include vitamins (e.g. vitamin A,vitamin B, vitamin C, vitamin D, vitamin E, etc.), minerals (e.g.,calcium, zinc, copper, manganese, chromium, molundenum, boron, etc),sugar (e.g., cellulose, dextrose, maltose, etc.), and/or naturalextracts (e.g., herb, ginseng, echinacea, green tea, glucosamine,omega-3, lutein, folic acid, liver oil, fish oil, coffee extracts,etc.). Formulations suitable for consumption by an individual (e.g., ahuman) include, without limitation, ready-to-mix powders, ready-to-drinkbeverages, functional shots, supplement tablets and capsules, coffeecreamers, bars, bites or baked goods, “no bone” broth, non-dairy frozennovelty, gummies (e.g., candy), chocolates, and meat snacks.Non-limiting examples of formulations containing the non-naturallyoccurring polypeptides are provided in Examples 4-6.

A composition, formulation, or product is “nutritional” or “nutritive”if it provides an appreciable amount of nourishment to its intendedconsumer, meaning the consumer assimilates all or a portion of thecomposition or formulation into a cell, organ, and/or tissue. Generally,such assimilation into a cell, organ, and/or tissue provides a benefitor utility to the consumer, e.g., by maintaining or improving the healthand/or natural function(s) of said cell, organ, and/or tissue. Anutritional composition or formulation that is assimilated as describedherein is termed “nutrition”. By way of non-limiting example, apolypeptide is nutritional if it provides an appreciable amount ofpolypeptide nourishment to its intended consumer, meaning the consumerassimilates all or a portion of the protein, typically in the form ofsingle amino acids or small peptides, into a cell, organ, and/or tissue.“Nutrition” also means the process of providing to a subject, such as ahuman or other mammal, a nutritional composition, formulation, product,or other material. A nutritional product need not be “nutritionallycomplete”, meaning if consumed in sufficient quantity, the productprovides all carbohydrates, lipids, essential fatty acids, essentialamino acids, conditionally essential amino acids, vitamins, and mineralsrequired for health of the consumer. Additionally, a “nutritionallycomplete protein” contains all protein nutrition required (meaning theamount required for physiological normalcy by the organism) but does notnecessarily contain micronutrients such as vitamins and minerals,carbohydrates, or lipids.

In preferred embodiments, a composition or formulation is nutritional inits provision of polypeptide capable of decomposition (e.g., thebreaking of a peptide bond, often termed protein digestion) to singleamino acids and/or small peptides (e.g., two amino acids, three aminoacids, or four amino acids, possibly up to ten amino acids) in an amountsufficient to provide a “nutritional benefit”. In addition, in certainembodiments provided are nutritional polypeptides that transit acrossthe gastrointestinal wall and are absorbed into the bloodstream as smallpeptides (e.g., larger than single amino acids but smaller than aboutten amino acids) or larger peptides, oligopeptides, or polypeptides(e.g., >11 amino acids). A nutritional benefit in apolypeptide-containing composition can be demonstrated and, optionally,quantified, by a number of metrics. For example, a nutritional benefitis the benefit to a consuming organism equivalent to or greater than atleast about 0.5% of a reference daily intake value of protein, such asabout 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%,about 8%, about 9%, about 10%, about 15%, about 20%, about 25%, about30%, about 35%, about 40%, about 45%, about 50%, about 60%, about 65%,about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about100% or greater than about 100% of a reference daily intake value.Alternatively, a nutritional benefit is demonstrated by the feelingand/or recognition of satiety by the consumer. In other embodiments, anutritional benefit is demonstrated by incorporation of a substantialamount of the polypeptide component of the composition or formulationinto the cells, organs, and/or tissues of the consumer, suchincorporation generally meaning that single amino acids or shortpeptides are used to produce polypeptides de novo intracellularly. A“consumer” or a “consuming organism” means any animal capable ofingesting the product having the nutritional benefit. Typically, theconsumer is a mammal such as a healthy human, e.g., a healthy infant,child, adult, or older adult. Alternatively, the consumer is a mammalsuch as a human (e.g., an infant, child, adult, or older adult) at riskof developing or suffering from a disease, disorder, or conditioncharacterized by (i) the lack of adequate nutrition and/or (ii) thealleviation thereof by the nutritional products of the presentdisclosure. An “infant” is generally a human under about age 1 or 2, a“child” is generally a human under about age 18, and an “older adult” or“elderly” human is a human aged about 65 or older.

Herein provided are nutritive polypeptides (e.g., non-naturallyoccurring polypeptides described herein) capable of transforming healthand treating, preventing and reducing the severity of a multitude ofdiseases, disorders, and conditions associated with amino acidpathophysiology, as they are selected for specific physiologic benefitsto improve health and address many nutrition-related conditions,including gastrointestinal malabsorption, muscle wasting, diabetes orpre-diabetes, obesity, oncology, metabolic diseases, and other cellularand systemic diseases. Also provided are the compositions andformulations that contain the nutritive polypeptides (e.g.,non-naturally occurring polypeptides described herein), as food,beverages, medical foods, supplements, and pharmaceuticals.

Nutritive polypeptides (e.g., non-naturally occurring polypeptidesdescribed herein) can be evaluated for their physicochemical andfunctional properties can be evaluated (see, e.g., Example 7). Suchproperties may include digestibility, allergenicity, thermostability,solubility, aggregation, toxicity, taste, and mouth/feelcharacteristics.

In some embodiments, the formulations are incorporated into foodproducts having advantages over similar food products lacking thenutritive polypeptides (e.g., non-naturally occurring polypeptidesdescribed herein), or the formulations are incorporated into otherproducts such as beverage products or animal feed products. For example,the food products have a reduced fat content, a reduced sugar content,and/or a reduced calorie content compared to a food product not havingthe nutritive polypeptide (e.g., non-naturally occurring polypeptidesdescribed herein). Preferably, the nutritive polypeptide (e.g.,non-naturally occurring polypeptides described herein) is present in thefood product such that consumption of a nutritional amount of the foodproduct is satiating. In an embodiment, gelatin, an animal-derivedmaterial, is replaced by a non-animal derived product, containing one ormore nutritive polypeptides (e.g., non-naturally occurring polypeptidesdescribed herein). Typically the nutritive polypeptide (e.g.,non-naturally occurring polypeptides described herein) is present in anamount effective to replace gelatin in the product. The gelatinreplacement is incorporated into a food product, a beverage product, oran animal feed product, and the formulation is substantially free ofnon-comestible products.

Also provided herein are formulations containing a nutritive polypeptide(e.g., non-naturally occurring polypeptides described herein) present ina functional and/or nutritional amount, which increases the viscosity ofa food or beverage product, such as formulations containingviscosity-increasing nutritive polypeptides (e.g., non-naturallyoccurring polypeptides described herein) incorporated into food productshaving advantages over similar food products lacking the nutritivepolypeptides (e.g., non-naturally occurring polypeptides describedherein). For example, the food products have a reduced fat content, areduced sugar content, and/or a reduced calorie content compared to afood product not having the nutritive polypeptide (e.g., non-naturallyoccurring polypeptides described herein). Viscous nutritive polypeptides(e.g., non-naturally occurring polypeptides described herein) can beused as a nutritionally favorable low calorie substitute for fat.Additionally, it may be desired to add to the compositions and productsone or more polysaccharides or emulsifiers, resulting in a furtherimprovement in the creamy mouthfeel.

In certain embodiments, the non-naturally occurring polypeptides of thedisclosure may be combined with other ingredients to provide combinationproducts. Such ingredients may include carbohydrates, lipids,supplemental minerals, supplemental vitamins, excipients or bufferingagents, flavoring agents, sweeteners, or coloring agents.

A “carbohydrate” refers to a sugar or polymer of sugars. The terms“saccharide”, “polysaccharide,” “carbohydrate”, and “oligosaccharide”can be used interchangeably. Most carbohydrates are aldehydes or ketoneswith many hydroxyl groups, usually one on each carbon atom of themolecule. Carbohydrates generally have the molecular formula CnH2nOn. Acarbohydrate can be a monosaccharide, a disaccharide, trisaccharide,oligosaccharide, or polysaccharide. The most basic carbohydrate is amonosaccharide, such as glucose, sucrose, galactose, mannose, ribose,arabinose, xylose, and fructose. Disaccharides are two joinedmonosaccharides. Exemplary disaccharides include sucrose, maltose,cellobiose, and lactose. Typically, an oligosaccharide includes betweenthree and six monosaccharide units (e.g., raffinose, stachyose), andpolysaccharides include six or more monosaccharide units. Exemplarypolysaccharides include starch, glycogen, and cellulose. Carbohydratesmay contain modified saccharide units such as 2′-deoxyribose wherein ahydroxyl group is removed, 2′-fluororibose wherein a hydroxyl group isreplace with a fluorine, or N-acetylglucosamine, a nitrogen-containingform of glucose (e.g., 2′-fluororibose, deoxyribose, and hexose).Carbohydrates may exist in many different forms, for example,conformers, cyclic forms, acyclic forms, stereoisomers, tautomers,anomers, and isomers.

As used herein a “lipid” includes fats, oils, triglycerides,cholesterol, phospholipids, fatty acids in any form including free fattyacids. Fats, oils and fatty acids can be saturated, unsaturated (cis ortrans) or partially unsaturated (cis or trans). In some embodiments thelipid comprises at least one fatty acid selected from lauric acid(12:0), myristic acid (14:0), palmitic acid (16:0), palmitoleic acid(16:1), margaric acid (17:0), heptadecenoic acid (17:1), stearic acid(18:0), oleic acid (18:1), linoleic acid (18:2), linolenic acid (18:3),octadecatetraenoic acid (18:4), arachidic acid (20:0), eicosenoic acid(20:1), eicosadienoic acid (20:2), eicosatetraenoic acid (20:4),eicosapentaenoic acid (20:5) (EPA), docosanoic acid (22:0), docosenoicacid (22:1), docosapentaenoic acid (22:5), docosahexaenoic acid (22:6)(DHA), and tetracosanoic acid (24:0). In some embodiments thecomposition comprises at least one modified lipid, for example a lipidthat has been modified by cooking.

Additional ingredients also include supplemental minerals or mineralsources. Examples of minerals include, without limitation: chloride,sodium, calcium, iron, chromium, copper, iodine, zinc, magnesium,manganese, molybdenum, phosphorus, potassium, and selenium. Suitableforms of any of the foregoing minerals include soluble mineral salts,slightly soluble mineral salts, insoluble mineral salts, chelatedminerals, mineral complexes, non-reactive minerals such as carbonylminerals, and reduced minerals, and combinations thereof.

Additional ingredients also include one or more supplemental vitamins.The vitamin can be fat-soluble or water soluble vitamins. Suitablevitamins include but are not limited to vitamin C, vitamin A, vitamin E,vitamin B12, vitamin K, riboflavin, niacin, vitamin D, vitamin B6, folicacid, pyridoxine, thiamine, pantothenic acid, and biotin. Suitable formsof any of the foregoing are salts of the vitamin, derivatives of thevitamin, compounds having the same or similar activity of the vitamin,and metabolites of the vitamin.

The formulations may also include excipients or buffering agents.Non-limiting examples of suitable excipients include a tastant, aflavorant, a buffering agent, a preservative, a stabilizer, a binder, acompaction agent, a lubricant, a dispersion enhancer, a disintegrationagent, a flavoring agent, a sweetener, a coloring agent. Non-limitingexamples of suitable buffering agents include sodium citrate, magnesiumcarbonate, magnesium bicarbonate, calcium carbonate, and calciumbicarbonate.

The formulations may also include a preservative. Non-limiting examplesof suitable preservatives include organic acids that are naturallyderived from fermentation (such as citric, ascorbic, propionic acids),antimicrobial peptides (nisin) or other suitable preservatives (such assalt, calcium sorbate, sodium sorbate).

Binding agents, lubricants, dispersion enhancers, disintegrants and thelike may also be used as an excipient. Non-limiting examples of suitablebinders include starches, pregelatinized starches, gelatin,polyvinylpyrolidone, cellulose, methylcellulose, sodiumcarboxymethylcellulose, ethylcellulose, polyacrylamides,polyvinyloxoazolidone, polyvinylalcohols, C12-C18 fatty acid alcohol,polyethylene glycol, polyols, saccharides, oligosaccharides, andcombinations thereof. Non-limiting examples of suitable lubricantsinclude magnesium stearate, calcium stearate, zinc stearate,hydrogenated vegetable oils, sterotex, polyoxyethylene monostearate,talc, polyethyleneglycol, sodium benzoate, sodium lauryl sulfate,magnesium lauryl sulfate, and light mineral oil. Non-limiting examplesof suitable dispersants include starch, alginic acid,polyvinylpyrrolidones, guar gum, kaolin, bentonite, purified woodcellulose, sodium starch glycolate, isoamorphous silicate, andmicrocrystalline cellulose as high HLB emulsifier surfactants.Non-limiting examples of suitable non-effervescent disintegrants includestarches such as corn starch, potato starch, pregelatinized and modifiedstarches thereof, sweeteners, clays, such as bentonite,micro-crystalline cellulose, alginates, sodium starch glycolate, gumssuch as agar, guar, locust bean, karaya, pecitin, and tragacanth. Insome embodiments the disintegrant is an effervescent disintegrant.Non-limiting examples of suitable effervescent disintegrants includesodium bicarbonate in combination with citric acid, and sodiumbicarbonate in combination with tartaric acid.

Additional ingredients may also include flavoring agents, sweeteners, orcoloring agents. Flavoring agents incorporated into the outer layer canbe chosen from synthetic flavor oils and flavoring aromatics; naturaloils; extracts from plants, leaves, flowers, and fruits; andcombinations thereof. Non-limiting examples of suitable sweetenersinclude glucose (corn syrup), dextrose, invert sugar, fructose, andmixtures thereof (when not used as a carrier); saccharin and its varioussalts such as the sodium salt; dipeptide sweeteners such as aspartame;dihydrochalcone compounds, glycyrrhizin; Stevia rebaudiana (Stevioside);chloro derivatives of sucrose such as sucralose; and sugar alcohols suchas sorbitol, mannitol, sylitol, and the like. Non-limiting examples ofsuitable color agents include food, drug and cosmetic colors (FD&C),drug and cosmetic colors (D&C), and external drug and cosmetic colors(Ext. D&C).

Solid dosage forms for oral administration include capsules, tablets,caplets, pills, troches, lozenges, powders, and granules. A capsuletypically comprises a core material comprising a protein or compositionand a shell wall that encapsulates the core material. In someembodiments, the core material comprises at least one of a solid, aliquid, and an emulsion. In some embodiments, the shell wall materialcomprises at least one of a soft gelatin, a hard gelatin, and a polymer.

Powders or granules embodying the polypeptides and compositionsdisclosed herein can be incorporated into a food product. In someembodiments the food product is a drink for oral administration.Non-limiting examples of a suitable drink include fruit juice, a fruitdrink, an artificially flavored drink, an artificially sweetened drink,a carbonated beverage, a sports drink, a liquid diary product, a shake,an alcoholic beverage, a caffeinated beverage, infant formula, and soforth. Other suitable means for oral administration include aqueous andnonaqueous solutions, creams, pastes, emulsions, suspensions andslurries, each of which may optionally also contain at least one ofsuitable solvents, preservatives, emulsifying agents, suspending agents,diluents, sweeteners, coloring agents, a tastant, a flavorant, andflavoring agents.

Suitable examples of a solid foodstuff include without limitation a foodbar, a snack bar, a cookie, a brownie, a muffin, a cracker, a biscuit, acream or paste, an ice cream bar, a frozen yogurt bar, and the like.

A formulation can contain a nutritive polypeptide (e.g., non-naturallyoccurring polypeptides as described herein) in an amount based on theconcentration of the nutritive polypeptide (e.g., non-naturallyoccurring polypeptides as described herein) (e.g., on a weight-to-weightbasis), such that the nutritive polypeptide (e.g., non-naturallyoccurring polypeptides as described herein) accounts for up to 100% ofthe weight of the formulation, meaning that all or essentially all ofthe matter present in the formulation is in the form of the nutritivepolypeptide (e.g., non-naturally occurring polypeptides as describedherein). More typically, about 99%, about 98%, about 97%, about 96%,about 95%, about 90%, about 85%, about 80%, about 75%, about 70%, about65%, about 60%, about 55%, about 50%, about 45%, about 40%, about 35%,about 30%, about 25%, about 20%, about 15%, about 10%, about 5% or lessthan about 5% of the weight present in the formulation is in the form ofthe nutritive polypeptide (e.g., non-naturally occurring polypeptides asdescribed herein). In some embodiments, the formulation contains 10 mg,100 mg, 500 mg, 750 mg, 1 g, 2 g, 3 g, 4 g, 5 g, 6 g, 7 g, 8 g, 9, 10 g,15 g, 20 g, 25 g, 30 g, 35 g, 40 g, 45 g, 50 g, 60 g, 70 g, 80 g, 90 g,100 g, or over 100 g of nutritive polypeptide.

In some embodiments, the polypeptides or compositions are provided in adosage form. In some embodiments, the dosage form is designed foradministration of at least one polypeptide disclosed herein, wherein thetotal amount of polypeptide administered is selected from 0.1 g to 1 g,1 g to 5 g, from 2 g to 10 g, from 5 g to 15 g, from 10 g to 20 g, from15 g to 25 g, from 20 g to 40 g, from 25 g to 50 g, and from 30 g to 60g. In some embodiments, the dosage form is designed for administrationof at least one protein disclosed herein, wherein the total amount ofprotein administered is selected from about 0.1 g, 0.1 g to 1 g, 1 g, 2g, 3 g, 4 g, 5 g, 6 g, 7 g, 8 g, 9 g, 10 g, 15 g, 20 g, 25 g, 30 g, 35g, 40 g, 45 g, 50 g, 55 g, 60 g, 65 g, 70 g, 75 g, 80 g, 85 g, 90 g, 95g, and 100 g.

In some embodiments the protein or composition is consumed at a rate offrom 0.1 g to 1 g a day, 1 g to 5 g a day, from 2 g to 10 g a day, from5 g to 15 g a day, from 10 g to 20 g a day, from 15 g to 30 g a day,from 20 g to 40 g a day, from 25 g to 50 g a day, from 40 g to 80 g aday, from 50 g to 100 g a day, or more.

In another aspect, this disclosure provides methods of maintaining orincreasing at least one of muscle mass, muscle strength, and functionalperformance in a subject. In some embodiments, the methods compriseproviding to the subject a sufficient amount of a polypeptide of thisdisclosure, a composition of this disclosure, or a composition made by amethod of this disclosure. In some embodiments, the subject is at leastone of elderly, critically-medically ill, and suffering fromprotein-energy malnutrition. In some embodiments, the sufficient amountof a polypeptide of this disclosure, a composition of this disclosure,or a composition made by a method of this disclosure is consumed by thesubject in coordination with performance of exercise. In someembodiments, the polypeptide of this disclosure, composition of thisdisclosure, or composition made by a method of this disclosure isconsumed by the subject by an oral, enteral, or parenteral route. Insome embodiments, the polypeptide of this disclosure, composition ofthis disclosure, or composition made by a method of this disclosure isconsumed by the subject by an oral route. In some embodiments, thepolypeptide of this disclosure, composition of this disclosure, orcomposition made by a method of this disclosure is consumed by thesubject by an enteral route.

In another aspect, this disclosure provides methods of maintaining orachieving a desirable body mass index in a subject. In some embodiments,the methods comprise providing to the subject a sufficient amount of apolypeptide of this disclosure, a composition of this disclosure, or acomposition made by a method of this disclosure. In some embodiments,the subject is at least one of elderly, critically-medically ill, andsuffering from protein-energy malnutrition. In some embodiments, thesufficient amount of a polypeptide of this disclosure, a composition ofthis disclosure, or a composition made by a method of this disclosure isconsumed by the subject in coordination with performance of exercise. Insome embodiments, the polypeptide of this disclosure, composition ofthis disclosure, or composition made by a method of this disclosure isconsumed by the subject by an oral, enteral, or parenteral route.

In another aspect, this disclosure provides methods of providing apolypeptide (e.g., of the disclosure) to a subject with protein-energymalnutrition. In some embodiments, the methods comprise providing to thesubject a sufficient amount of a polypeptide of this disclosure, acomposition of this disclosure, or a composition made by a method ofthis disclosure. In some embodiments, the polypeptide of thisdisclosure, composition of this disclosure, or composition made by amethod of this disclosure is consumed by the subject by an oral,enteral, or parenteral route.

The polypeptides of this disclosure are useful for treating sarcopeniaor frailty once it develops in a subject or for preventing the onset ofsarcopenia or frailty in a subject who is a member of an at risk groups.In some embodiments, all of the polypeptide consumed by the subject is apolypeptide according to this disclosure. In some embodiments,polypeptides according to this disclosure are combined with othersources of protein and/or free amino acids to provide the total proteinintake of the subject. In some embodiments, the subject is at least oneof elderly, critically-medically ill, and suffering from protein-energymalnutrition. In some embodiments, the polypeptide according todisclosure, the composition according to disclosure, or the compositionmade by a method according to disclosure is consumed by the subject incoordination with performance of exercise. In some embodiments, thepolypeptide according to this disclosure, the composition according todisclosure, or the composition made by a method according to disclosureis consumed by the subject by an oral, enteral, or parenteral route.

In some embodiments, incorporating at least one polypeptide orcomposition of this disclosure into the diet of a subject has at leastone effect selected from inducing postprandial satiety (including bysuppressing hunger), inducing thermogenesis, reducing glycemic response,positively affecting energy expenditure positively affecting lean bodymass, reducing the weight gain caused by overeating, and decreasingenergy intake. In some embodiments, incorporating at least onepolypeptide or composition of this disclosure into the diet of a subjecthas at least one effect selected from increasing loss of body fat,reducing lean tissue loss, improving lipid profile, and improvingglucose tolerance and insulin sensitivity in the subject.

In some embodiments, the composition including the non-naturallyoccurring polypeptides (e.g., recombinant proteins and/or purifiedrecombinant proteins) can be formulated for topical application. Thetopical application can be for medical purpose or cosmetic purpose. Insuch embodiments, the composition may further include at least one of acarrier molecule (e.g., vehicle), a preservative, and/or additionaledible ingredients. Any suitable carrier molecules are contemplated, andthe exemplary carrier molecule may include water, oil, alcohol,propylene glycol, or emulsifiers. In addition, any suitablepreservatives are contemplated, and the exemplary preservatives includezinc oxide, parabens, formaldehyde releasers, isothiazolinones,phenoxyethanol, or organic acids such as benzoic acid, sodium benzoate,or butylene glycol, hexanediol, or potassium sorbate.

In one aspect, the compositions that comprise non-naturally occurringpolypeptides may be personal care products (e.g., a cosmetic). In someembodiments, the compositions are formulated for topical administration.The compositions can contain other cosmetic ingredients suitable forhuman use. The personal care products may be useful for preventing ortreating ultraviolet radiation damage to human skin or hair. Thepersonal care products may be useful for increasing the firmness,elasticity, brightness, hydration, tactile texture or visual texture ofskin and/or stimulate collagen production. The personal care productsmay be useful for reducing redness of the skin. The personal careproducts may be applied to skin or hair. The compositions include, forexample, masks, skin cleaners such as soap, cleansing creams, cleansinglotions, facial cleansers, cleansing milks, cleansing pads, facialwashes, facial and body creams and moisturizers, facial serums, facialand body masks, facial toners and mists, eye creams and eye treatments,exfoliator formulas, lip balms and lipsticks, hair shampoo, hairconditioner and body shampoos, hair and scalp serums, hair mists andsprays, eye shadow, concealer, mascara and other color cosmetics.

The compositions that comprise the non-naturally occurring polypeptidecan further comprise at least one additional ingredient comprising atopical carrier or a preservative. The topical carrier may comprise atopical carrier selected from the group consisting of liposome,biodegradable microcapsule, lotion, spray, aerosol, dusting powder,biodegradable polymer, mineral oil, triglyceride oil, silicone oil,glycerin, glycerin monostearate, alcohols, emulsifying agents, liquidpetroleum, white petrolatum, propylene glycol, polyoxyethylene,polyoxypropylene, wax, sorbitan monostearate, polysorbate, cetyl esterwax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol, cyclomethicone,cyclopentasiloxane and water. The preservative may comprise apreservative selected from the group consisting of tocopherol,diiodomethyl-p-tolylsulfone, 2-Bromo-2-nitropropane-1,3-diol, cis isomer1-(3-chloroallyl)-3,5,7-triaza-1-azoniaadamantane chloride,glutaraldehyde, 4,4-dimethyl oxazolidine, 7-Ethylbicyclooxazolidine,phenoxyethanol, butylene glycol, 1,2 Hexanediol, methyl paraben, sorbicacid, Germaben® II, rosemary extract, and EDTA

Also provided in certain embodiments herein, are methods of decreasingskin damage, promoting the repair of damaged skin, protecting skinagainst UV damage, and/or protecting skin cells against the effects ofexposure to urban dust. In another embodiment, methods of increasing thefirmness, elasticity, brightness, hydration, tactile texture, or visualtexture of skin and/or stimulating collagen production are provided. Themethods may comprise a step of applying a composition comprising anon-naturally occurring polypeptide of the disclosure to the skin of asubject. Without being bound to a particular theory or mechanism, thenon-naturally occurring polypeptide in the composition may decrease skindamage by protecting against UV damage. In some cases, the non-naturallyoccurring polypeptide in the composition may promote the repair ofdamaged skin by increasing the viability of cells. In some cases, thenon-naturally occurring polypeptide in the composition may decrease skindamage and/or promote repair of cells by increasing procollagensynthesis when applied to skin, and/or promoting the viability of skincells. In some cases, the non-naturally occurring polypeptide decreasesthe formation of thymine-thymine (TT) dimer formation.

The methods provided herein encompass the use of a composition fortreatment indicated in the method, such as by the steps provided herein.In embodiments, the disclosure provides the use of a compositionprovided herein (e.g., a non-naturally occurring polypeptide or aformulation comprising a non-naturally occurring polypeptide) in amethod for decreasing skin damage, promoting the repair of damaged skin,protecting skin against UV damage, and/or protecting skin cells againstthe effects of exposure to urban dust (e.g., such as by administering tothe skin of a subject a composition provided herein). In embodiments,the disclosure provides the use of a composition provided herein (e.g.,a non-naturally occurring polypeptide or a formulation comprising anon-naturally occurring polypeptide) in a method for increasing thefirmness, elasticity, brightness, hydration, tactile texture, or visualtexture of skin and/or stimulating collagen production.

Provided in certain embodiments herein are (e.g., topical) compositionsor formulations comprising one or more non-naturally occurringpolypeptide provided herein (e.g., for cosmetic use). In someembodiments, the composition provides any suitable amount of polypeptideprovided herein, such as in any suitable amount (e.g., an amountsuitable to provide a benefit when given or administered to anindividual or cell). In some specific embodiments, the compositioncomprises an amount suitable to provide a beneficial effect to the skinof an individual when (e.g., topically) administered to the skin of theindividual. In specific embodiments, the composition comprises between0.001% and 30% w/w of a polypeptide (or non-naturally occurring collagenpolypeptide) such as provided herein. In more specific embodiments, thecomposition comprises between 0.001% and 20% w/w of a polypeptide (ornon-naturally occurring collagen polypeptide) such as provided herein,between 0.001% and 10% w/w of a polypeptide (or non-naturally occurringcollagen polypeptide) such as provided herein, between 0.001% and 5% w/wof a polypeptide (or non-naturally occurring collagen polypeptide) suchas provided herein, between 0.001% and 2% w/w of a polypeptide (ornon-naturally occurring collagen polypeptide) such as provided herein,between 0.001% and 1% w/w of a polypeptide (or non-naturally occurringcollagen polypeptide) such as provided herein, between 0.001% and 0.5%w/w of a polypeptide (or non-naturally occurring collagen polypeptide)such as provided herein, and between 0.001% and 0.2% w/w of apolypeptide (or non-naturally occurring collagen polypeptide) such asprovided herein.

In various embodiments, the concentration or amount of a non-naturallyoccurring polypeptide (e.g., recombinant protein) provided herein is ina composition provided herein in any suitable amount and may, e.g., varydepending on the use or formulation (e.g., gel, capsule, liquid, powder,etc.). Exemplary concentrations of the non-naturally occurringpolypeptides (e.g., recombinant proteins) in the composition can be atleast about 0.01%, at least about 0.05%, at least about 0.1%, at leastabout 0.2%, at least about 0.5%, at least about 1%, at least about 5%,at least about 10%, at least about 15%, at least about 20%, at leastabout 25%, at least about 30%, at least about 35%, at least about 40%,at least about 45%, at least about 50%, at least about 55%, at leastabout 60%, at least about 65%, at least about 70%, at least about 75%,at least about 80%, at least about 85%, at least about 90%, at leastabout 95%, at least about 98% (w/v or w/w) in the composition.Alternatively and/or additionally, the exemplary concentration of thenon-naturally occurring polypeptides (e.g., recombinant proteins) in thecomposition can be about 0.01%, about 0.05%, about 0.1%, about 0.2%, atleast 0.5%, 1%, about 5%, about 10%, about 15%, about 20%, about 25%,about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%,about 95%, about 98% (w/v or w/w) in the composition. Alternativelyand/or additionally, the exemplary concentration of the non-naturallyoccurring polypeptides (e.g., recombinant proteins) in the compositioncan range from about 0.01% to about 99%, from about 0.05% to about 99%,from about 0.1% to about 99%, from about 0.1% to about 99%, from about0.5% to about 99%, from about 0.1% to about 10%, from about 1% to about99%, from about 5% to about 99%, from about 10% to about 99%, from about15% to about 99%, from about 20% to about 99%, from about 25% to about99%, from about 30% to about 99%, from about 35% to about 99%, fromabout 40% to about 99%, from about 45% to about 99%, from about 50% toabout 99%, from about 55% to about 99%, from about 60% to about 99%,from about 65% to about 99%, from about 70% to about 99%, from about 75%to about 99%, from about 80% to about 99%, from about 85% to about 99%,from about 90% to about 99%, from about 95% to about 99%, from about0.1% to about 90%, from about 1% to about 90%, from about 5% to about90%, from about 10% to about 90%, from about 15% to about 90%, fromabout 20% to about 90%, from about 25% to about 90%, from about 30% toabout 90%, from about 35% to about 90%, from about 40% to about 90%,from about 45% to about 90%, from about 50% to about 90%, from about 55%to about 90%, from about 60% to about 90%, from about 65% to about 90%,from about 70% to about 90%, from about 75% to about 90%, from about 80%to about 90%, from about 85% to about 90%, from about 20% to about 80%,from about 25% to about 80%, from about 30% to about 80%, from about 35%to about 80%, from about 40% to about 80%, from about 45% to about 80%,from about 50% to about 80%, from about 55% to about 80%, from about 60%to about 80%, from about 65% to about 80%, from about 70% to about 80%,from about 75% to about 80%, from about 70% to about 99%, from about 75%to about 99%, from about 80% to about 99%, etc. Alternatively and/oradditionally, the exemplary concentration of the non-naturally occurringpolypeptides (e.g., recombinant proteins) in the composition can be lessthan about 95%, about 90%, about 85%, about 80%, about 75%, about 70%,about 65%, about 60%, about 55%, about 50%, about 45%, about 40%, etc.

Certain aspects of the disclosure include methods of improving theappearance of the skin, the hair, and/or the nails of a subject byadministering the composition to the subject. Additionally and/oralternatively, the disclosure includes a method of improving the jointhealth and/or restoring bone density in a subject. In some embodiments,the subject has or is suspected to have osteoporosis and/orosteoarthritis. Alternatively and/or additionally, the disclosureincludes a method of improving gut health, altering or improving themicrobiome of a subject, or altering and/or reducing inflammation ortissue repair in a subject. In some embodiments, the composition isadministered orally in a dose and schedule sufficient or effective forimproving the appearance of the skin, the hair, and/or the nails of asubject, improving the joint health and/or restoring bone density in asubject having osteoporosis and/or osteoarthritis, and/or improving guthealth, altering or improving the microbiome of a subject, or alteringand/or reducing inflammation or tissue repair in a subject. Any suitabledose is optionally used. In some embodiments, the dose used is fromabout 0.1 mg/kg to about 200 mg/kg, from about 0.2 mg/kg to about 150mg/kg, from about 0.5 mg/kg to about 150 mg/kg, from about 0.5 mg/kg toabout 100 mg/kg, from about 0.8 mg/kg to about 100 mg/kg, from about 1.0mg/kg to about 100 mg/kg, from about 1.0 mg/kg to about 90 mg/kg, fromabout 1.0 mg/kg to about 80 mg/kg, from about 1.0 mg/kg to about 70mg/kg, from about 1.0 mg/kg to about 60 mg/kg, from about 1.0 mg/kg toabout 50 mg/kg, about 0.5 mg/kg, about 1 mg/kg, about 1.5 mg/kg, about2.0 mg/kg, about 2.5 mg/kg, about 3.0 mg/kg, about 3.5 mg/kg, about 4.0mg/kg, about 4.5 mg/kg, or about 5.0 mg/kg, about 10 mg/kg, about 15mg/kg, about 20 mg/kg, about 25 mg/kg, or about 30 mg/kg. In someembodiments, the dose may increase or decrease by the schedule of theadministration. For example, the dose for administering to a subject(e.g., human) can be increased or decreased for about 1 mg/kg, about 2mg/kg, about 3 mg/kg, about 4 mg/kg, about 5 mg/kg, about 6 mg/kg, about7 mg/kg, about 8 mg/kg, about 9 mg/kg, or about 1.0 mg/kg per eachadministration (e.g., for 3 consecutive administration, the dose can beincreased from 2.0 mg/kg, 2.2 mg/kg, 2.4 mg/kg, respectively, etc.). Inanother example, the dose for administering to a subject (e.g., human)can be increased and then decreased, or decreased and then increased forabout 1 mg/kg, about 2 mg/kg, about 3 mg/kg, about 4 mg/kg, about 5mg/kg, about 6 mg/kg, about 7 mg/kg, about 8 mg/kg, about 9 mg/kg, orabout 1.0 mg/kg per each administration (e.g., for 5 consecutiveadministration, the dose can be increased from 2.0 mg/kg, 2.2 mg/kg, 2.4mg/kg, then 2.2 mg/kg, and 2.0 mg/kg, respectively, etc.).

In some embodiments, the schedule of administration varies depending onthe purpose, gender, age, or health condition of the subject. Forexample, in some embodiments, the composition is administered once aday, twice a day, three times a day, up to 6 times a day, every 2 days,every 3 days, every 4 days, every 5 days, every 6 days, etc.Alternatively and/or additionally, in some embodiments, the compositionis administered a plurality of times in an irregular interval, orincreased interval, or decreased interval.

In certain embodiments, the composition is topically applied in a doseand/or schedule sufficient or effective for improving the appearance ofthe skin, the hair, and/or the nails of a subject, and/or reducinginflammation in a subject. In some instances, the dose varies dependingon the target area for the topical application (e.g., hair, skin, wound,nail, etc.), and can range from about 0.1 g/inch² to about 10 g/inch²,from about 0.1 g/inch² to about 9 g/inch², from about 0.1 g/inch² toabout 8 g/inch², from about 0.1 g/inch² to about 7 g/inch², from about0.1 g/inch² to about 6 g/inch², from about 0.1 g/inch² to about 5g/inch², from about 0.1 g/inch² to about 4 g/inch², from about 0.1g/inch² to about 3 g/inch², from about 0.5 g/inch² to about 5 g/inch²,from about 0.5 g/inch² to about 4 g/inch², from about 1 g/inch² to about4 g/inch², or from about 1 g/inch² to about 3 g/inch², of area fortopical application. In certain embodiments, the dose varies dependingon the purpose, gender, age, severity of damage on the area, or healthcondition. For example, the composition can be applied to the targetarea of the subject at least three times a day, at least twice a day,once a day, up to 6 times a day, every 2 days, every 3 days, every 4days, every 5 days, every 6 days, etc.

Skin appearance and quality: In some embodiments, provided herein aremethods of improving skin appearance and/or quality, such as byadministering an effective amount of a product or composition (e.g.,containing a non-naturally occurring polypeptide described herein)provided herein to an individual (e.g., an individual in need of suchimprovement). In some embodiments, administration is orally ortopically. In some instances, administration results in various changesor effects on the skin of the individual. In some instances, the skin ofthe individual demonstrates increased proliferation and/or reduced celldeath rate (e.g., when tested using a colorimetric assay for assessingcell metabolic activity (e.g., MTT assay)). In some embodiments, theskin demonstrates improved production of extracellular matrix (ECM)components such as collagen, elastin, fibronectin, fibrillin and/ordecreased production of matrix-degrading proteins (e.g., matrixmetalloproteinases (MMPs) and proteases). In certain instances, the skinshows resistance or improved outcome upon exposure to harmful agentslike photodamage (e.g., UV irradiation), pollution (e.g., urban dust),and/or harsh skincare actives (e.g., retinoic acid, benzoyl peroxide,salicylic acid). In certain instances, such resistance or improvedoutcome is shown via improved cell viability or proliferation (orreduced cell death) that can be assessed using MTT viability assay, viaimproved DNA repair that can be assessed by thymidine-dimer ELISAdetection, reduced inflammation that can be assessed by Luminexdetection, reduced reactive oxidative stress (ROS) that can be assessedby CM-H₂DCFDA (General oxidative stress indicator) detection.

In some instances, the skin demonstrates reduction in wrinkles and/orfine lines, reduction in skin redness and/or hyperpigmentation, increasein skin brightness, decrease in pore size, decrease in skin roughness,and/or reduction in acne (e.g., when assessed using CLARITY analysis).In certain instances, the skin demonstrates improvement in skinelasticity, increase in skin firmness, increase in skin hydration,increase in skin barrier function, increase in skin collagen and elastincontent, and/or increase in dermal density.

Hair Quality: In some embodiments, provided herein are methods ofimproving hair appearance and/or quality, such as by administering aneffective amount of a product or composition provided herein (e.g.,containing a non-naturally occurring polypeptide as described herein) toan individual (e.g., an individual in need of such improvement). In someembodiments, administration is orally or topically. In some instances,administration results in various changes or effects on the hair of theindividual. In certain instances, the hair demonstrates improved hairfiber thickness and/or density, increases in moisture, faster growthrate, reduced split ends, reduced frizz/increased static control,improved fiber alignment/shine, increased combability, and/or strongerresistance to hair breakage. In some instances, the hair demonstratesimproved hair growth, thicker hair fiber diameter, increasedcombability, reduced hair loss, and/or increased hair tensile strength.

Nail Quality: In some embodiments, provided herein are methods ofimproving nail appearance and/or quality, such as by administering aneffective amount of a product or composition provided herein (e.g.,containing a non-naturally occurring polypeptide as described herein) toan individual (e.g., an individual in need of such improvement). In someembodiments, administration is orally or topically. In some instances,administration results in various changes or effects on the nail of theindividual. In certain instances, the nail demonstrates improved(reductions in) nail peeling, nail edge irregularities and/or nailroughness, frequency of cracked/chipped nails, and/or increases in nailgrowth rate.

Joint health: In some embodiments, provided herein are methods ofimproving joint health, such as by administering an effective amount ofa product or composition provided herein (e.g., containing anon-naturally occurring polypeptide as described herein) to anindividual (e.g., an individual in need of such improvement). In someembodiments, administration is orally. In some instances, administrationresults in various changes or effects in joint health of the individual.In certain instances, the improved joint health is demonstrated byreduction in reported joint pain and/or increase in range of jointmobility.

Inflammation: In some embodiments, provided herein are methods ofimproving inflammatory effects, such as by administering an effectiveamount of a product or composition provided herein (e.g., containing anon-naturally occurring polypeptide as described herein) to anindividual (e.g., an individual in need of such improvement). In someembodiments, administration is orally or topically. In some instances,administration results in various changes or effects in inflammation inthe individual. In certain instances, the improved inflammatory effectis demonstrated by lower cytokine levels in the bloodstream (e.g.,assessed by Luminex detection) and/or restore healthy levels of immunecells (e.g., by blood differential counts).

Gut health: In some embodiments, provided herein are methods ofimproving gut health, such as by administering an effective amount of aproduct or composition provided herein (e.g., containing a non-naturallyoccurring polypeptide as described herein) to an individual (e.g., anindividual in need of such improvement). In some embodiments,administration is orally. In some instances, administration results invarious changes or effects in gut health of the individual. In certaininstances, the improved gut health is demonstrated by improved bowelmovements and/or decrease in gastrointestinal discomfort/pain.

Microbiome: In some embodiments, provided herein are methods of alteringand/or improving the microbiome, such as by administering an effectiveamount of a product or composition provided herein (e.g., containing anon-naturally occurring polypeptide as described herein) to anindividual (e.g., an individual in need of such improvement). In someembodiments, administration is orally. In some instances, administrationresults in various changes or effects in the microbiome of theindividual. In certain instances, the improved microbiome isdemonstrated by increased diversity of microbes and/or increasedabundance of beneficial microbes (e.g., assessed by 16S DNA sequencingstool samples).

EXAMPLES Example 1. Generation of Non-Naturally Occurring Polypeptidesof the Disclosure

This example shows the generation of a recombinant polypeptide of thedisclosure by genetically engineered microorganisms and purificationprocess of such generated polypeptides.

The polynucleotides of SEQ ID NOs: 1, 3, 5, and 7 were synthesized andat least one of the polynucleotides were inserted into a pET vector.Overlaps between a pET vector and SEQ ID NOs: 1, 3, 5, and 7 weredesigned to be between 20 and 30 bp long and added using PCR with theenzyme PRIMESTAR® GXL polymerase(www.takarabio.com/products/per/gc-rich-per/primestar-gxl-dna-polymerase).The opened pET vector and insert DNA (e.g., polynucleotide of SEQ IDNO: 1) were assembled together into the final plasmid using IN-FUSION®Cloning (www.takarabio.com/products/cloning/in-fusion-cloning). In allcases, the nucleic acid sequences were preceded by a secretion signalsequence disclosed as SEQ ID NOs: 9, 11, 13, 15, 17, 19, 21, or 23.Plasmid sequences were verified through Sanger sequencing.

Cells were transformed with final plasmids and subsequently cultivatedin minimal media and frozen in 1.5 aliquots with vegetable glycerin at aratio of 50:50 of cells to glycerin. One vial of this frozen culture wasrevived in 50 ml of minimal media overnight at 37° C., 200 rpm.Formulations of the minimal media in this example are shown in Table 2and Table 3. Cells were then transferred into 300 ml of minimal mediaand grown for 6-9 hours to reach an optical density (OD) 600 of 5-10.

The fermentations were performed at various temperature ranging from 25°to 28° C. For some fermentations, the temperature of the fermentationwas maintained at a constant temperature and immediately upon completionof fermentation the polypeptide was purified. For other fermentations,the temperature of the fermentations was maintained for a desired periodof time and when cell densities of OD600 of 10-20 were reached, thetemperature was reduced to induce protein production. Typically, thetemperature was reduced from 28° C. to 25° C. Induction was carried outby adding IPTG to the media at concentrations ranging from 0.1-0.5 mM.Fermentations were continued for 40-60 hours.

The recombinant polypeptide was purified as follows: The pH of thefermentation broth was decreased to between 3-3.5 using 5-50% SulfuricAcid. The cells were then separated using centrifugation orcentrifugation followed by microfiltration. Supernatant of the acidifiedbroth was tested on a polyacrylamide gel and found to containrecombinant polypeptide in relatively high abundance compared tostarting pellet. To obtain volume and salt reduction, concentration anddiafiltration steps were performed ultrafiltration. Final polypeptideslurry was run on an SDS-PAGE gel to confirm presence of the recombinantpolypeptide.

To verify that the desired proteins were produced, supernatants fromcultures of microbes carrying SEQ ID NOs: 1, 3, 5, or 7 were collectedand purified by decreasing their pH as described above. The acidifiedbroth was analyzed by SDS-PAGE, and bands corresponding to the expectedsize protein were detected in relative purity. As shown in FIG. 1, athick and clear band was observed at the expected sizes for eachrespective protein. Samples were subsequently analyzed for quantifyingrecombinant polypeptide titers and purity by reverse phase and sizeexclusion HPLC chromatography and mass spectrometry, which confirmed thecorrect identity of the respective proteins of interest.

FIGS. 2A-2C depict SDS-PAGE gels of non-naturally occurring polypeptidesof the disclosure before and after treatment at pH 3.0. FIG. 2A depictsan SDS-PAGE gel of fermentation supernatant containing a non-naturallyoccurring polypeptide having an amino acid sequence of SEQ ID NO: 2before (Lane 1) and after (Lane 2) treatment at pH 3.0. The expectedmolecular weight of such polypeptide was about 17.9 kDa. The identity ofthe polypeptide was confirmed by mass spectrometry (data not shown).FIG. 2B depicts an SDS-PAGE gel of fermentation supernatant containing anon-naturally occurring polypeptide having an amino acid sequence of SEQID NO: 8 before (Lane 3) and after (Lane 4) treatment at pH 3.0. Theexpected molecular weight of such polypeptide was about 17.6 kDa. Theidentity of the polypeptide was confirmed by mass spectrometry (data notshown). FIG. 2C depicts an SDS-PAGE gel of fermentation supernatantcontaining a non-naturally occurring polypeptide produced in variousbacterial host strains having an amino acid sequence of SEQ ID NO: 8before (Lanes 3-5) and after (Lanes 6-8) treatment at pH 3.0.

Example 2. Human Clinical Study of the Non-Naturally OccurringPolypeptides of the Disclosure

Skin appearance and quality: Patients are recruited and/or culturedhuman skin cells or patient-derived skin samples are provided toevaluate the benefit of recombinant polypeptides provided herein.Non-naturally occurring polypeptides as described herein (or productscontaining non-naturally occurring polypeptides as described herein) andcontrol products are administered (orally or topically) to separate (invitro or in vivo) cohorts to evaluate for various effects on skin. Skineffects are evaluated quantitatively and/or qualitatively. For example,when the composition including the non-naturally occurring polypeptideis applied to or administered to, the cultured human skin cells in vitro(either primary culture or cell line), or human skin tissue ex vivo, thecultured human skin cells or cells in the human skin tissue showincreased proliferation or reduced cell death rate (e.g., when testedusing a colorimetric assay for assessing cell metabolic activity (e.g.,MTT assay)). In some instances, such cultured human skin cells or cellsin the human skin tissue contacted or treated with the compositionsincluding the non-naturally occurring polypeptides described herein mayshow, via RNA-seq transcriptomic analysis or proteomics analysis,increased production of extracellular matrix (ECM) components such ascollagen, elastin, fibronectin, fibrillin, and decreased production ofmatrix-degrading proteins (e.g., matrix metalloproteinases (MMPs) andproteases). Such treated cultured human skin cells or cells in the humanskin tissue are evaluated to demonstrate resistance or improved outcomeupon exposure to harmful agents like photodamage (e.g., UV irradiation),pollution (e.g., urban dust), and harsh skincare actives (e.g., retinoicacid, benzoyl peroxide, salicylic acid). Such resistance or improvedoutcome is shown via improved cell viability or proliferation (orreduced cell death) that is assessed using MTT viability assay, viaimproved DNA repair that is assessed by thymidine-dimer ELISA detection,reduced inflammation that is assessed by Luminex detection, and/orreduced reactive oxidative stress (ROS) that is assessed by CM-H₂DCFDA(General oxidative stress indicator) detection.

In another example, when the composition including a non-naturallyoccurring polypeptide (e.g., as described herein) is applied to, oradministered to the subject orally or topically on the skin, thesubject's skin is evaluated for reduction in wrinkles and fine lines,reduction in skin redness and hyperpigmentation, increase in skinbrightness, decrease in pore size, decrease in skin roughness, andreduction in acne (e.g., when assessed using CLARITY analysis). The skinis also further evaluated (before and after administration) to showchange in skin elasticity, change in skin firmness, change in skinhydration, change in skin barrier function, change in skin collagen andelastin content, and/or change in dermal density.

Hair Quality: Effects of products (e.g., containing a non-naturallyoccurring polypeptide as described herein) provided herein (e.g.,relative to control products) on hair are also evaluated. For example,when the products provided herein (e.g., containing a non-naturallyoccurring polypeptide as described herein) are applied to hair or orallyadministered to a subject, hair quality is measured, such as bymeasuring changes in hair fiber thickness and density, changes inmoisture, changes in growth rate, changes in prevalence of split ends,changes in frizz/increased static control, changes in fiberalignment/shine, changes in combability, and/or changes in resistance tohair breakage (e.g., measured by in vitro hair tress testing). In someinstances, clinical testing measures changes in hair growth, hair fiberdiameter, combability, hair loss, and/or hair tensile strength.

Nail Quality: Effects of products (e.g., containing a non-naturallyoccurring polypeptide as described herein) provided herein (e.g.,relative to control products) on nails are also evaluated. For example,when the products provided herein (e.g., containing a non-naturallyoccurring polypeptide as described herein) are applied to nail or orallyadministered to a subject, nail quality is measured, such as bymeasuring changes in nail hardness, nail peeling, nail edgeirregularities and nail roughness, frequency of cracked/chipped nails,and/or nail growth rate.

Joint health: Effects of products (e.g., containing a non-naturallyoccurring polypeptide as described herein) provided herein (e.g.,relative to control products) on joints are also evaluated. For example,when the products provided herein (e.g., containing a non-naturallyoccurring polypeptide as described herein) are orally administered to asubject, joint quality is measured, such as by measuring changes inreported joint pain and/or range of joint mobility.

Inflammation: Effects of products (e.g., containing a non-naturallyoccurring polypeptide as described herein) provided herein (e.g.,relative to control products) on inflammation are also evaluated. Forexample, when the products provided herein (e.g., containing anon-naturally occurring polypeptide as described herein) are orallyadministered to a subject, changes in inflammation are measured,cytokine levels in the bloodstream are measured (e.g., assessed byLuminex detection), and/or levels of immune cells are measured (by blooddifferential counts).

Gut health: Effects of products (e.g., containing a non-naturallyoccurring polypeptide as described herein) provided herein (e.g.,relative to control products) in gut health are also evaluated. Forexample, when the products provided herein (e.g., containing anon-naturally occurring polypeptide as described herein) are orallyadministered to a subject, changes in bowel movements and/orgastrointestinal discomfort/pain are measured.

Microbiome: Effects of products (e.g., containing a non-naturallyoccurring polypeptide described herein) provided herein (e.g., relativeto control products) in the microbiome are also evaluated. For example,when the products provided herein (e.g., containing a non-naturallyoccurring polypeptide described herein) are orally administered to asubject, changes in diversity of microbes or abundance of beneficialmicrobes is measured, which can be assessed by 16S DNA sequencing stoolsamples. Also, such effect can be shown in vitro as the compositionsupports growth of beneficial microbes in broth co-cultures.

Example 3. In Vitro Studies of Non-Naturally Occurring Polypeptides ofthe Disclosure

This example demonstrates functional effects on cells in vitro aftertreatment with a non-naturally occurring polypeptide having the aminoacid sequence of SEQ ID NO: 2.

A Non-Naturally Occurring Polypeptide of SEQ ID NO: 2 IncreasesViability of Human Dermal Fibroblasts.

Human primary fibroblasts were cultured in media alone (FIG. 3; “A”), orwith 0.025% w/w (FIG. 3; “B”), 0.05% w/w (FIG. 3; “C”), or 0.1% w/w(FIG. 3; “D”) of a non-naturally occurring polypeptide having the aminoacid sequence of SEQ ID NO: 2 for 24 hours. Cell viability was evaluatedusing the MTT colorimetric assay. As shown in FIG. 3, fibroblaststreated with the polypeptide of SEQ ID NO: 2 showed an increase in cellviability relative to the media only control.

A Non-Naturally Occurring Polypeptide of SEQ ID NO: 2 Increases CollagenType I Production in Human Dermal Fibroblasts.

Human primary fibroblasts were cultured in media alone (FIG. 4; “A”), orwith 0.025% w/w (FIG. 4; “B”), 0.05% w/w (FIG. 4; “C”), or 0.1% w/w(FIG. 4; “D”) of a non-naturally occurring polypeptide having the aminoacid sequence of SEQ ID NO: 2 for 24 hours. Fibroblast production ofcollagen type I was determined by analyzing the supernatants with anenzyme-linked immunosorbent assay (ELISA) for pro-collagen type IC-peptide, which is a readout for total secreted collagen type I. Asshown in FIG. 4, fibroblasts treated with the polypeptide of SEQ ID NO:2 secreted higher levels of collagen type I than media control-treatedfibroblasts.

A Non-Naturally Occurring Polypeptide of SEQ ID NO: 2 Increases CollagenType I Production in Human Tenocytes.

Human primary tenocytes were cultured in media alone (FIG. 5; “A”), orwith 0.025% w/w (FIG. 5; “B”) or 0.05% w/w (FIG. 5; “C”) of anon-naturally occurring polypeptide having the amino acid sequence ofSEQ ID NO: 2 for 24 hours. Tenocyte production of collagen type I wasdetermined by analyzing the supernatants with an enzyme-linkedimmunosorbent assay (ELISA) for pro-collagen type I C-peptide, which isa readout for total secreted collagen type I. As shown in FIG. 5,tenocytes treated with the polypeptide of SEQ ID NO: 2 secreted higherlevels of collagen type I than media control-treated cells.

Example 4. Sports Drink Containing a Non-Naturally Occurring Polypeptideof the Disclosure

In this example, a non-naturally occurring polypeptide of the disclosurewas formulated in a sports drink.

Sports Drink Formulation:

10 g polypeptide of SEQ ID NO: 2/12 oz serving

Ingredients Listing:

Water, collagen peptides, sugar, tangerine juice concentrate, salt,citric acid, monopotassium phosphate, sodium citrate, fruit andvegetable juice [for color], natural flavor, Stevia

Variables Tested:

1. 10 g vs. 12 g polypeptide of SEQ ID NO: 2/12 oz serving

2. 0%-15% fruit juice concentrate

3. 7 g-20 g sugar/12 oz serving

4. Sweetener systems: sucrose, monk fruit, Stevia

5. 0.05%-0.30% citric acid

Example 5. Gummies Containing a Non-Naturally Occurring Polypeptide ofthe Disclosure

In this example, a non-naturally occurring polypeptide of the disclosurewas formulated in a gummy.

2.5 g polypeptide of SEQ ID NO: 2 & 100 mg hyaluronic acid/25 g serving

Ingredients Listing:

Tapioca syrup, cane sugar, water, collagen peptides, citric acid,pectin, sodium citrate, natural flavor, sodium hyaluronate, fruit andvegetable juice [for color]

Variables Tested:

1. Order of addition—polypeptide of SEQ ID NO: 2 needs to be made into asolution and added after the syrup cooking step

2. Various levels of polypeptide of SEQ ID NO: 2 (2%, 6%, 8%, 10%)

3. Polypeptide of SEQ ID NO: 2 has a buffering effect; tested differentcitrate/citric acid levels.

Example 6. Brownies Containing a Non-Naturally Occurring Polypeptide ofthe Disclosure

In this example, a non-naturally occurring polypeptide of the disclosurewas formulated in a brownie.

3 g polypeptide of SEQ ID NO: 2/40 g serving

Ingredients Listing:

(Bread) flour, cane sugar, cocoa powder, water, coconut oil, polypeptideof SEQ ID NO: 2, olive oil, glycerin, vanilla extract, baking soda,salt, xanthan, lecithin.

Variables Tested:

1. All-purpose flour vs. bread flour

2. Polypeptide of SEQ ID NO: 2 at 3 g, 3.75 g, 5 g, or 9 g/40 g serving

3. Reduced sugar 20%, 30%

Example 7. Properties of Non-Naturally Occurring Polypeptides of theDisclosure Related to Nutritional Use

In this example, the non-naturally occurring polypeptide of SEQ ID NO: 2was evaluated for various properties related to nutritional use.

Viscosity

The non-naturally occurring polypeptides provided herein can beevaluated for viscosity in solution. In this example, a polypeptide ofSEQ ID NO: 2 was shown to be soluble up to 43% w/w at pH 4.5 and 50% w/wat pH 6.5, using a flow sweep on DHR-II rheometer with 40 mm parallelplate at 25° C. A polypeptide of SEQ ID NO: 2 as a spray dried powderwas found to go into solution slower in water at 50° C. or higher versuswater at ambient temperature. Results are depicted in FIG. 7A and FIG.7B.

Interactions of a polypeptide of SEQ ID NO: 2 with hydrocolloids andoils was also evaluated. Blends of a polypeptide of SEQ ID NO: 2 and gumarabic were prepared in DI water and evaluated for viscosity in theratios according to Table 4.

TABLE 4 Ratios of gum arabic and polypeptide blends Gum arabic SEQ IDNO: 2 20% 0  20% 10% 10%  5% 10% 0  20% 20% 10% 10% 10% 10% but pH 6.5

Blends of a polypeptide of SEQ ID NO: 2 and xanthan were prepared in DIwater and evaluated for viscosity in various ratios. FIG. 8 depictsresults of this study. SDA represents a polypeptide of SEQ ID NO: 2spray dried at pH 6.5. SDB represents a polypeptide of SEQ ID NO: 2spray dried with a feed of 20% solids at pH 4.5.

Gel Hardness

The non-naturally occurring polypeptides of the disclosure can beevaluated for gel hardness in solution. Briefly, 5% protein solutions(containing a polypeptide of SEQ ID NO: 2) were crosslinked with 100 utransglutaminase enzyme. Protein and enzyme mixtures were deposited in12-well cell plates and incubated at 50° C. for 2 hours. Gels wereheated to 100° C. for 10 minutes to inactivate transglutaminase enzyme.Gels were cooled at ambient temperature and stored in 4° C. overnight.Gel hardness was evaluated by molding 4 mL of gel mixture in 23 mmdiameter wells. Gels were brought to ambient temperature prior tomeasurements, and hardness of gel was recorded as the force at which ½″stainless steel ball probe (TA-18) was depressed 2 mm into the gel at 1mm/sec using TA.XT Plus Texture Analyzer instrument. Protein solutionswere prepared in DI water or 10 mM sodium phosphate buffer, pH 7.2.Solutions were adjusted to target pH using 1M HCl or 2N NaOH prior toaddition of enzyme. Results of this study are depicted in FIG. 9.

Protein solutions were prepared in DI water or 10 mM sodium phosphatebuffer, pH 7.2. Solutions were adjusted to target pH using 1M HCl or 2NNaOH prior to addition of enzyme. Results are depicted in FIG. 10A andFIG. 10B. At pH 5.5, crosslinked GL21 gels range in hardness from 27-35g. Between pH 6.3-6.4, crosslinked polypeptide gels range in hardnessfrom 20-31 g.

Emulsion Properties

The non-naturally occurring polypeptides described herein can beevaluated for emulsion properties in solution. Protein solutions at pH4.5 were mixed with canola oil at a 5:1 ratio and were homogenized usingIKA Ultra Turrax at 10000 rpm for 10 min. Stability of emulsion wasevaluated after 24 hours at ambient temperature in 12 mL conical tubes.

Foamability and Foam Stability

The non-naturally occurring polypeptides described herein can beevaluated for foaming properties in solution. 10 mL of 5% w/w of apolypeptide of SEQ ID NO: 2 solution from various lots was shaken in aconical 50 mL tube for 2 minutes. Volume of foam and time of foamcollapse were recorded, and results are depicted in Table 5.

TABLE 5 Volume of foam and time of foam collapse. Approximate volumeTime to foam Lot number foam generated (mL) collapse (min) benchmark 275.5 PP6-GL21-20-016 35 >25 PP6-GL21-20-030 15 1.5 PP6-GL21-20-048 25 20PP7-GL21-20-028 25 >25 PP7-GL21-20-036 30 >25 PP7-GL21-20-175 12 14PP5-GL21-20-265 15 10 PP5-GL21-20-293 0 — PP5-GL21-20-307 10 >25PP5-GL21-20-321 25 2

Sensory Notes

The non-naturally occurring polypeptides of the disclosure can beevaluated for sensory properties, including odor and flavor. Spray driedpolypeptide of SEQ ID NO: 2 from various lots was evaluated either driedor in solution, and the results are depicted in Table 6.

TABLE 6 Sensory notes. Odor 4.2% Flavor 4.2% Color 4.2% Odor w/w w/w w/wLot number powder solution solution solution PP7-GL21- Slight gelatinAcidic, tart. Clear, slight 20-028 aroma/milk Slightly yellow hintprotein astringent. but not as concentrate Some dairy/ much as any (MPC)grassy flavor of the other products PP7-GL21- Strong Dairy/ acidic,Light yellow 20-036 MPC/ cheesy aftertaste barnyard PP5-GL21- Light MPCDairy/ — Clear 20-265 cheesy PP5-GL21- Medium Dairy/ — Clear 20-293 MPCcheesy PP5-GL21- Light MPC Clean — Clear 20-307 PP5-GL21- Sweet, lightClean, — Clear 20-321 MPC sweet PP5-GL21- Light MPC Clean, faint — Clear20-335 dairy, sweet PPS-GL21- Light MPC Dairy/ — Clear 20-337WB cheesy

Solubility

Non-naturally occurring polypeptides of the disclosure can be evaluatedfor solubility. The effect of agglomeration with lecithin solution oncompacted powder forms of a polypeptide of SEQ ID NO: 2 to increaseparticle size and improve solubility was evaluated. 2.5 g protein powderwas dropped into 50 mL water. Wettability was evaluated by observingsinking within 20 seconds. Dissolution was evaluated with 40 seconds ofslow stirring and 60 seconds of rest. Results are depicted in Table 7.

TABLE 7 Solubility of a polypeptide of SEQ ID NO: 2. Particle CompactedCompacted with Lecithin Mesh size (um) Yield (%) Wet Dissolve Yield (%)Wet Dissolve  >18 >1000  1% — —  2% — — 20-18  850-1000 13% Y N 22% Y N25-20 710-850 22% Y N 18% Y N 30-25 600-710 12% Y N 13% Y N 35-30500-600 12% Y N 11% Y N 40-35 425-500  7% Y Y  7% Y Y 60-40 250-425 23%N Y 16% Y Y 80-60 180-250  8% N N  7% Y Y 140-80  106-180  1% — —  4% NY <140  <106  3% — — — —

Viscosity of solutions of a polypeptide of SEQ ID NO: 2 at 20% w/wsolutions were also determined at 25° C., and results are depicted inFIG. 11.

8% w/w protein solution (containing a polypeptide of SEQ ID NO: 2) at pH5.5 and 100 u transglutaminase enzyme was homogenized with 50% w/w oilat 26000 rpm with Polytron for 1 minute on ice. 4 g of mixture wasdeposited into each well of a 12-well cell plate and incubated for 2hours at 50° C. Gels were heated to 100° C. for 10 minutes to inactivatethe enzyme. Gels were cooled at ambient temperature and stored at 4° C.overnight. A polypeptide of SEQ ID NO:2 stabilized high oil emulsionduring crosslinking reaction to form protein oil gel. Results aredepicted in FIG.

Example 8. Polypeptide Sequence Confirmation of Products and Lack ofHydroxyproline Residues

Mass spectrometry was used to confirm the sequence of a polypeptide ofSEQ ID NO: 2 produced by methods according to this disclosure. Table 8and Table 9 provide the results of peptide mapping of this polypeptide.

TABLE 8 Peptide mapping of the polypeptide of SEQ ID NO: 2. Table 8 discloses SEQ ID NOS: 35-77,respectively, in order of appearance.  Calculated Observed MassRetention Se- Mass  Mass  Error  Time  Intensity Label quence Range (Da)(Da) (Da) (min) (counts) T1- DTGFP 1 10 1049.4601 1049.4598 −0.0002167.49 84,835.78 Mox GMPGR T1- DTGFP 1 13 1292.5455 1292.5471 0.0013739.13 126,314.00 2- GMPGR clipD SGD T1- DTGFP 1 16 1602.7209 1602.7206−0.00029 8.12 577,210.30 2 GMPGR SGDPG R T1- DTGFP 1 19 1874.86941874.8634 −0.005825 7.19 1,090,023.00 3 GMPGR SGDPG RSGK T1- DTGFP 1 292830.3457 2830.3513 0.005645 8.6 6,936.58 4 GMPGR SGDPG RSGKD GLPGS PGFKT1- TGFPG 2 10 918.43817 918.43848 0.000225 8.38 4,768.16 clipT MPGR T1-GFPGM 3 19 1658.7947 1658.7977 0.003076 7.19 228,217.00 3- PGRSG clipGDPGRS GK T1- PGMPG 5 19 1454.7048 1454.7067 0.001883 7.19 436,082.90 3-RSGDP clipP2 GRSGK T1- PGRSG 8 19 1169.5901 1169.5885 −0.001693 7.1925,095.66 3- DPGRS clipP1 GK T2 SGDPG 11 16 588.2736 Not detected R T2-SGDPG 11 29 1814.8911 1814.8878 −0.003236 6.62 1,121.89 4 RSGKD GLPGSPGFK T3 SGK 17 19 291.1663 Not detected T4 DGLPG 20 29 974.4941Not detected SPGFK T4- DGLPG 20 31 1159.5509 1159.551 3.39E−05 8.583,738.19 5- SPGFK clipE GE T4- DGLPG 20 44 2431.188 2431.197 0.0086498.48 112,201.00 5 SPGFK GEVGQ PGSPG LEGHR T4- DGLPG 20 59 3803.89793803.9045 0.00648 9.39 441,091.10 6 SPGFK GEVGQ PGSPG LEGHR GEPGI PGIPGNQGAK T4- DGLPG 20 62 4117.0728 4117.0747 0.002786 8.83 114,096.20 7SPGFK GEVGQ PGSPG LEGHR GEPGI PGIPG NQGAK GQK T4- GLPGS 21 59 3688.87113688.8735 0.002824 9.01 60,853.30 6- PGFKG clipG EVGQP GSPGL EGHRG EPGIPGIPGN QGAK T4- PGSPG 23 44 2146.0557 2146.0562 0.000427 8.47 14,717.965- FKGEV clipP GQPGS PGLEG HR T5 GEVGQ 30 44 1476.7189 Not detectedPGSPG LEGHR T5- GEVGQ 30 59 2848.4216 2848.4216 −5.61E−05 8.28 71,435.916 PGSPG LEGHR GEPGI PGIPG NQGAK T5- GEVGQ 30 62 3161.5967 3161.59860.00197 7.68 14,067.67 7 PGSPG LEGHR GEPGI PGIPG NQGAK GQK T5- RGEPG 4459 1546.8215 1546.822 0.00052 7.68 3,424.51 6- IPGIP clipR GNQGA K T6GEPGI 45 59 1391.7277 Not detected PGIPG NQGAK T6- GEPGI 45 62 1703.89551703.8969 0.001454 7.58 13,208.12 7 PGIPG NQGAK GQK T6- GEPGI 45 742777.4824 2777.48 −0.002657 9.09 24,234.60 8 PGIPG NQGAK GQKGE IGPPGLPGAK T6- GEPGI 45 98 4955.5239 4955.5317 0.008078 10.72 9,805.94 9PGIPG NQGAK GQKGE IGPPG LPGAK GSPGE TGLMG PEGSF GLPGA PGPK T6- PGNQG 5362 983.51483 983.51324 −0.001589 8.83 4,871.83 7- AKGQK clipP T6- PGNQG53 74 2057.1018 2057.1055 0.003865 9.41 1,757.15 8- AKGQK clipP GEIGPPGLPG AK T7 GQK 60 62 332.1928 Not detected T7- GQKGE 60 74 1418.78821418.79 0.002054 8.04 3,103.51 8- IGPPG MetI LPGAK T7- KGEIG 62 741219.6925 1219.691 −0.001366 7.46 11,881.92 8- PPGLP clipK GAK T8 GEIGP63 74 1092.6047 Not detected PGLPG AK T8- GEIGP 63 116 4920.47174920.4771 0.005323 9.79 81,370.18 11 PGLPG AKGSP GETGL MGPEG SFGLP GAPGPKGDKG EPGLQ GKPGS SGAK T8- IGPPG 65 74 905.53345 905.53308 −0.00042 8.7128,037.06 clipI LPGAK T9- GSPGE 75 98 2234.0081 2233.9998 −0.00825711.12 48,341.35 cation TGLMG PEGSF GLPGA PGPK T9 GSPGE 75 98 2197.0593Not detected TGLMG PEGSF GLPGA PGPK T9- GSPGE 75 100 2368.1006 2368.1003−0.000254 11.24 10,231.43 10- TGLMG clipD PEGSF GLPGA PGPKG D T9- GSPGE75 101 2496.1956 2496.1887 −0.006757 10.11 99,126.63 10 TGLMG PEGSFGLPGA PGPKG DK T9- TGLMG 80 98 1768.8818 1768.88 −0.001801 11.1 5,679.79clipT PEGSF GLPGA PGPK T9- GLMGP 81 98 1667.8341 1667.8339 −0.00031 11.11,873.69 clipG EGSFG LPGAP GPK T9- SFGLP 88 98 1026.5498 1026.5485−0.00134 8.97 904.06 clipS GAPGP K T10- GDKGE 99 116 1668.8431 1668.8383−0.004932 5.46 475,309.00 11 PGLQG KPGSS GAK T11- KGEPG 101 1161496.7947 1496.7939 −0.000828 5.39 5,921.06 11- LQGKP clipK GSSGA K T11GEPGL 102 116 1369.707 Not detected QGKPG SSGAK T11- GEPGL 102 1433839.908 3839.9158 0.007516 7.99 15,528.34 13 QGKPG SSGAK GEPGG PGAPGEPGYP GIPGT QGIKG DK T12 GEPGG 117 140 2189.0752 2189.0723 −0.0030839.65 145,576.30 PGAPG EPGYP GIPGT QGIK T12- GEPGG 117 143 2489.21882489.219 0.000101 8.74 45,482.75 13 PGAPG EPGYP GIPGT QGIKG DK T12-PGEPG 125 154 2923.4424 2923.4473 0.005102 7.56 18,260.02 14- YPGIPclipP4 GTQGI KGDKG SQGES GIQGR T12- PGYPG 128 154 2640.3257 2640.32620.000495 7.58 16,118.65 14- IPGTQ clipP3 GIKGD KGSQG ESGIQ GR T12- PGIPG131 154 2323.188 2323.1851 −0.0029 8.33 16,247.45 14- TQGIK clipP2 GDKGSQGESG IQGR T12- PGTQG 134 154 2056.0298 2056.0305 0.000825 7.5624,994.64 14- IKGDK clipP1 GSQGE SGIQG R T12- PGTQG 134 155 2056.02982056.0308 0.00072 8.33 13,644.56 15- IKGDK clipP GSQGE SGIQG RK T13 GDK141 143 319.1612 Not detected T13- GDKGS 141 154 1374.6488 1374.65080.001875 5.23 66,051.58 14 QGESG IQGR T13- GDKGS 141 155 1374.64881374.6508 0.001875 5.23 66,051.58 15 QGESG IQGRK T13- GDKGS 141 1581816.9027 1816.899 −0.00382 2.88 2,494.46 16 QGESG IQGRK GEK T13- GDKGS141 160 2030.0253 2030.022 −0.003288 2.72 2,147.75 17 QGESG IQGRK GEKGRT13- KGSQG 143 154 1202.6003 1202.5985 −0.00199 3.44 2,745.49 14- ESGIQclipK GR T14 GSQGE 144 154 1075.5126 Not detected SGIQG R T14- GSQGE 144155 1202.6003 1202.5985 −0.00199 3.44 2,745.49 15 SGIQG RK T14- GSQGE144 158 1516.7594 1516.7587 −0.000831 3.02 1,968.24 16 SGIQG RKGEK T14-GSQGE 144 160 1729.882 1729.8776 −0.00443 2.79 2,134.92 17 SGIQG RKGEKGR T15 K 155 155 147.1128 Not detected T15- KGEKG 155 173 1981.04531981.0386 −0.006898 5.56 19,503.56 18 RQGNP GLQGT EGLR T15- KGEKG 155179 2609.3269 2609.3267 −0.000342 5.51 112,503.40 19 RQGNP GLQGT EGLRGEQGEK T16- GEKGR 156 173 1852.9503 1852.9458 −0.004281 5.76 1,739.51 18QGNPG LQGTE GLR T16- GEKGR 156 182 2795.3911 2795.3877 −0.0033 5.518,967.10 20 QGNPG LQGTE GLRGE QGEKG EK T17 GR 159 160 232.1404Not detected T17- GRQGN 159 172 1382.6902 1382.6887 −0.001582 7.5812,756.85 18- PGLQG clipL TEGL T17- GRQGN 159 173 1538.7914 1538.79190.000687 6.31 25,249.43 18 PGLQG TEGLR T17- GRQGN 159 179 2167.0732167.0701 −0.002904 5.83 106,768.80 19 PGLQG TEGLR GEQGE K T17- GRQGN159 182 2481.2319 2481.2329 0.000921 5.59 36,064.79 20 PGLQG TEGLR GEQGEKGEK T17- GRQGN 159 184 2653.2805 2653.2791 −0.001494 5.66 10,397.58 21-PGLQG clipD TEGLR GEQGE KGEKG D T18 QGNPG 161 173 1326.676 Not detectedLQGTE GLR T18- QGNPG 161 179 1936.9238 1936.9227 −0.001056 7.6814,110.98 19 LQGTE GLRGE QGEK T18- QGNPG 161 182 2268.1094 2268.11010.000698 5.98 60,817.64 20 LQGTE GLRGE QGEKG EK T18- QGNPG 161 1842440.158 2440.1602 0.002185 6.14 17,499.44 21- LQGTE clipD GLRGE QGEKGEKGD T18- RGEQG 173 188 1711.8601 1711.8606 9.05E−05 5.06 7,889.07 21-EKGEK clipR GDPGI R T19 GEQGE 174 179 647.2995 Not detected K T19- GEQGE174 188 1555.759 1555.7548 −0.00416 5.3 24,352.07 21 KGEKG DPGIR T20 GEK180 182 333.1768 Not detected T20- GEKGD 180 188 927.47742 927.47589−0.001604 5.25 156,253.20 21 PGIR T21 GDPGI 183 188 614.3256Not detected R 

TABLE 9Peptide mapping of the polypeptide of SEQ ID NO: 2. Table 9 discloses SEQ ID NOS: 78112,respectively, in order of appearance.  Calculated Observed MassRetention Se- Mass Mass Error Time Intensity Label quence Range (Da)(Da) (Da) (min) (counts) T11- GEPG 102 143 3839.908 3839.9158 0.0075167.99  15,528.34 13 LQGK PGSS GAKG EPGG PGAP GEPG YPGI PGTQ GIKG DK T12GEPG 117 140 2189.0752 2189.0723 −0.003083 9.65 145,576.30 GPGA PGEPGYPG IPGT QGIK T12- GEPG 117 143 2489.2188 2489.219 0.000101 8.74 45,482.75 13 GPGA PGEP GYPG IPGT QGIK GDK T12- PGEP 125 154 2923.44242923.4473 0.005102 7.56  18,260.02 14- GYPG clipP4 IPGT QGIK GDKG SQGESGIQ GR T12- PGYP 128 154 2640.3257 2640.3262 0.000495 7.58  16,118.6514- GIPG clipP3 TQGI KGDK GSQG ESGI QGR T12- PGIP 131 154 2323.1882323.1851 −0.0029 8.33  16,247.45 14- GTQG clipP2 IKGD KGSQ GESG IQGRT12- PGTQ 134 154 2056.0298 2056.0305 0.000825 7.56  24,994.64 14- GIKGclipP1 DKGS QGES GIQG R T12- PGTQ 134 155 2056.0298 2056.0308 0.000728.33  13,644.56 15- GIKG clipP DKGS QGES GIQG RK T13 GDK 141 143319.1612 Not detected T13- GDKG 141 154 1374.6488 1374.6508 0.0018755.23  66,051.58 14 SQGE SGIQ GR T13- GDKG 141 155 1374.6488 1374.65080.001875 5.23  66,051.58 15 SQGE SGIQ GRK T13- GDKG 141 158 1816.90271816.899 −0.00382 2.88   2,494.46 16 SQGE SGIQ GRKG EK T13- GDKG 141 1602030.0253 2030.022 −0.003288 2.72   2,147.75 17 SQGE SGIQ GRKG EKGR T13-KGSQ 143 154 1202.6003 1202.5985 −0.00199 3.44   2,745.49 14- GESG clipKIQGR T14 GSQG 144 154 1075.5126 Not detected ESGI QGR T14- GSQG 144 1551202.6003 1202.5985 −0.00199 3.44   2,745.49 15 ESGI QGRK T14- GSQG 144158 1516.7594 1516.7587 −0.000831 3.02   1,968.24 16 ESGI QGRK GEK T14-GSQG 144 160 1729.882 1729.8776 −0.00443 2.79   2,134.92 17 ESGI QGRKGEKG R T15 K 155 155 147.1128 Not detected T15- KGEK 155 173 1981.04531981.0386 −0.006898 5.56  19,503.56 18 GRQG NPGL QGTE GLR T15- KGEK 155179 2609.3269 2609.3267 −0.000342 5.51 112,503.40 19 GRQG NPGL QGTE GLRGEQGE K T16- GEKG 156 173 1852.9503 1852.9458 −0.004281 5.76   1,739.5118 RQGN PGLQ GTEG LR T16- GEKG 156 182 2795.3911 2795.3877 −0.0033 5.51  8,967.10 20 RQGN PGLQ GTEG LRGE QGEK GEK T17 GR 159 160 232.1404Not detected T17- GRQG 159 172 1382.6902 1382.6887 −0.001582 7.58 12,756.85 18- NPGL clipL QGTE GL T17- GRQG 159 173 1538.7914 1538.79190.000687 6.31  25,249.43 18 NPGL QGTE GLR T17- GRQG 159 179 2167.0732167.0701 −0.002904 5.83 106,768.80 19 NPGL QGTE GLRG EQGE K T17- GRQG159 182 2481.2319 2481.2329 0.000921 5.59  36,064.79 20 NPGL QGTE GLRGEQGE KGEK T17- GRQG 159 184 2653.2805 2653.2791 −0.001494 5.66 10,397.58 21- NPGL clipD QGTE GLRG EQGE KGEK GD T18 QGNP 161 1731326.676 Not detected GLQG TEGL R T18- QGNP 161 179 1936.9238 1936.9227−0.001056 7.68  14,110.98 19 GLQG TEGL RGEQ GEK T18- QGNP 161 1822268.1094 2268.1101 0.000698 5.98  60,817.64 20 GLQG TEGL RGEQ GEKG EKT18- QGNP 161 184 2440.158 2440.1602 0.002185 6.14  17,499.44 21- GLQGclipD TEGL RGEQ GEKG EKGD T18- RGEQ 173 188 1711.8601 1711.8606 9.05E−055.06   7,889.07 21- GEKG clipR EKGD PGIR T19 GEQG 174 179 647.2995Not detected EK T19- GEQG 174 188 1555.759 1555.7548 −0.00416 5.3 24,352.07 21 EKGE KGDP GIR T20 GEK 180 182 333.1768 Not detected T20-GEKG 180 188 927.47742 927.47589 −0.001604 5.25 156,253.20 21 DPGI R T21GDPG 183 188 614.3256 Not detected IR

Analysis was also performed to evaluate any amino acid or peptidemodifications present in the produced polypeptide of SEQ ID NO: 2.(Table 10). In a few instances, additional confirmatory analyses wereperformed to differential methionine oxidation from the presence ofhydroxyproline residues. For example, based upon the fragmentationresults from MS/MS scans, the tryptic peptide T1 (sequence DTGFPGMPGR(SEQ ID NO: 35)) was shown to contain a methionine oxidation rather thana proline hydroxylation. Based on such results, it was conclusivelydetermined that tryptic peptide 1 (T1) has oxidation at methionineposition 7 and no evidence of hydroxyproline at position 5 or 8.Similarly, where there is another methionine in position 83 in trypticpeptide 9 (T9), there were no detectable levels of methionine oxidation,hydroxyproline in positions 77, 85, 92, 95, and 97, or hydroxylysine atposition 98 of the polypeptide. Accordingly, the truncated collagenpolypeptides of the present disclosure also differ from naturallyoccurring collagen polypeptides in their lack of hydroxyprolineresidues.

TABLE 10 Analysis of amino acid and peptide modifications of thepolypeptide of SEQ ID NO: 2. Relative Intensity Instensity ModificationsLabel Sequence (counts) (%) Oxidation Met, Missed T1 Oxidation (M)84,835.78 3.29 Cleavages, N and Clippped (T) 4,768.16 0.18 C terminalClips T1-2 Missed Cleavage, Clippped (D) 126,314.00 4.90 Missed Cleavage577,210.30 22.38 T1-3 2 Missed Cleavages 1,090,023.00 42.26 2 MissedCleavages, Clipped (G) 228,217.00 8.85 2 Missed Cleavages, Clipped (P1)436,082.90 16.91 2 Missed Cleavages, Clipped (P2) 25,095.66 0.97 T1-4 3Missed Cleavages 6,936.58 0.27 Missed Cleavages, N T4 Unmodified233,635.20 23.83 and C Terminal T4-5 Missed Cleavgae, Clipped (E)3,738.19 0.38 Clips Missed Cleavage 112,201.00 11.45 T4-6 2 MissedCleavages 441,091.10 44.99 2 Missed Cleavages, Clipped (P) 14,717.961.50 T4-7 3 Missed Cleavages 114,096.20 11.64 3 Missed Cleavages,Clipped (G) 60,853.30 6.21 Missed Cleavages, N T5 Unmodified 4,628.281.63 Terminal Clip T5-6 Missed Cleavage 71,435.91 25.11 Missed Cleavage,Clipped (R) 3,424.51 1.20 T6 Unmodified 177,768.80 62.48 T5-7 2 MissedCleavages 14,067.67 4.94 T6-7 Missed Cleavage 13,208.12 4.64 MissedClevages, N T6-8 2 Missed Cleavages 24,234.60 26.78 Terminal Clips, T6-93 Missed Cleavages 9,805.94 10.83 Methyl Ile, T7-8 Missed Cleavage,Clipped (P) 4,871.83 5.38 Dehydrated Gln Missed Cleavage, Methyl (I)3,103.51 3.43 Missed Cleavage, Clipped (K) 11,881.92 13.13 T7-9 2 MissedCleavages, Clipped (P) 1,757.15 1.94 T8 Dehydrated (E) 6,818.71 7.53Clipped (I) 28,037.06 30.98 Cation K, Missed T9 Cation K 48,341.35 19.07Cleavgaes, N Terminal Clipped (T) 5,679.79 2.24 Clips Clipped (G)1,873.69 0.74 Clipped (S) 904.06 0.36 T9-10 Missed Cleavage 99,126.6339.11 Missed Cleavage, Clipped (D) 10,231.43 4.04 T10-11 MissedCleavage, Clipped (K) 5,921.06 2.34 T8-11 3 Missed Cleavages 81,370.1832.11 Missed Cleavages T11 Unmodified 40,875.16 72.47 T11-13 2 MissedCleavages 15,528.34 27.53 Missed Cleavages, N T12 Unmodified 145,576.3051.93 Terminal Clips T12-13 Missed Cleavage 45,482.75 16.23 T12-14 2Missed Cleavages, Clipped (P4) 18,260.02 6.51 2 Missed Cleavages,Clipped (P3) 16,118.65 5.75 2 Missed Cleavages, Clipped (P2) 16,247.455.80 2 Missed Cleavages, 24,994.64 8.92 T12-15 3 Missed Cleavages,Clipped (P) 13,644.56 4.87 Missed Cleavages T13-14 Missed Cleavage66,051.58 36.91 T13-16 3 Missed Cleavages 2,494.46 1.39 T13-17 4 MissedCleavages 2,147.75 1.20 T14 Unmodified 101,400.00 56.67 T14-15 MissedCleavages 2,745.49 1.53 T14-17 3 Missed Cleavages 2,134.92 1.19 T14-16 2Missed Cleavages 1,968.24 1.10

While preferred embodiments of the present disclosure have been shownand described herein, it will be obvious to those skilled in the artthat such embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the disclosure. It should beunderstood that various alternatives to the embodiments of thedisclosure described herein may be employed in practicing theembodiments of the disclosure. It is intended that the following claimsdefine the scope of the disclosure and that methods and structureswithin the scope of these claims and their equivalents be coveredthereby.

What is claimed is:
 1. A non-naturally occurring polypeptide comprisingan amino acid sequence having at least 95% sequence identity to atruncate of SEQ ID NO: 32, wherein the truncate of SEQ ID NO: 32 has aC-terminal truncation of 50 amino acids to 650 amino acids relative toSEQ ID NO:
 32. 2. The non-naturally occurring polypeptide of claim 1,comprising the amino acid sequence of the truncate of SEQ ID NO:
 32. 3.The non-naturally occurring polypeptide of claim 1, wherein the truncateof SEQ ID NO: 32 further comprises an N-terminal truncation of 50 aminoacids to 750 amino acids.
 4. The non-naturally occurring polypeptide ofclaim 1, wherein the non-naturally occurring polypeptide does notcomprise one or more of: a laminin G domain, a Von Willebrand factortype A (vWA) domain, and a fibrillar collagen C-terminal domain.
 5. Thenon-naturally occurring polypeptide of claim 1, wherein thenon-naturally occurring polypeptide comprises one or more collagentriple helix repeats.
 6. The non-naturally occurring polypeptide ofclaim 1, wherein the non-naturally occurring polypeptide is 50 aminoacids to 250 amino acids in length.
 7. The non-naturally occurringpolypeptide of claim 1, comprising or consisting of an amino acidsequence having at least 80% sequence identity to the amino acidsequence of SEQ ID NO:
 8. 8. The non-naturally occurring polypeptide ofclaim 1, comprising or consisting of the amino acid sequence of SEQ IDNO:
 8. 9. The non-naturally occurring polypeptide of claim 1, whereinthe non-naturally occurring polypeptide is monomeric.
 10. Thenon-naturally occurring polypeptide of claim 1, wherein thenon-naturally occurring polypeptide does not form a stable triple helixstructure of a naturally occurring collagen.
 11. The non-naturallyoccurring polypeptide of claim 1, wherein fewer than 10% of prolinespresent in the non-naturally occurring polypeptide are hydroxylated. 12.The non-naturally occurring polypeptide of claim 1, wherein thenon-naturally occurring polypeptide comprises less than 5 wt. %glycosylation.
 13. A composition comprising a non-naturally occurringpolypeptide comprising an amino acid sequence having at least 80%sequence identity to a truncate of SEQ ID NO: 32, wherein the truncateof SEQ ID NO: 32 has an N-terminal truncation of 50 amino acids to 750amino acids, a C-terminal truncation of 50 amino acids to 650 aminoacids, or both the N-terminal truncation and the C-terminal truncation,relative to SEQ ID NO: 32, wherein the composition is a productformulated for consumption by an individual.
 14. The composition ofclaim 13, wherein the product is a food, a beverage, or a nutraceuticalsupplement, and wherein the non-naturally occurring polypeptide is aningredient present in the product in a concentration of at least 0.1%w/w.
 15. The composition of claim 13, wherein the product is a powder,and wherein the non-naturally occurring polypeptide is at least 50% w/wof the powder.
 16. The composition of claim 13, wherein the product is anutritive polypeptide product formulated as a food, a beverage, amedical food, a supplement, a pharmaceutical, or any combinationthereof.
 17. The composition of claim 13, wherein the product provides adosage of from 1 g to 5 g of the non-naturally occurring polypeptide.18. The composition of claim 13, wherein the product provides a dosageof from 2 g to 10 g of the non-naturally occurring polypeptide.
 19. Thecomposition of claim 13, wherein the composition is a food ingredient.20. The composition of claim 19, further comprising at least one of acarbohydrate, a lipid, a supplemental mineral, a supplemental vitamin,an excipient, a buffering agent, a flavoring agent, a sweetener, or acoloring agent.
 21. A method of improving the appearance of the skin,the hair, and/or the nails of a subject, the method comprisingadministering to the subject the composition of claim 13, therebyimproving the appearance of the skin, the hair, and/or the nails of thesubject, relative to prior to the administering.
 22. The method of claim21, wherein the administering comprises orally administering to thesubject.
 23. A method of improving gut health in a subject, the methodcomprising administering to the subject the composition of claim 13,thereby improving gut health in the subject, relative to prior to theadministering.
 24. The method of claim 23, wherein the administeringcomprises orally administering to the subject.
 25. A method of alteringor improving the microbiome in a subject, the method comprisingadministering to the subject the composition of claim 13, therebyaltering or improving the microbiome in the subject, relative to priorto the administering.
 26. A method of altering and/or reducinginflammation in a subject, the method comprising administering to thesubject the composition of claim 13, thereby altering and/or reducinginflammation in the subject, relative to prior to the administering. 27.A method of improving tissue repair in a subject, the method comprisingadministering to the subject the composition of claim 13, therebyimproving tissue repair in the subject, relative to prior to theadministering.
 28. A method of improving bone, muscle, and/or jointhealth in a subject, the method comprising administering to the subjectthe composition of claim 13, thereby improving bone, muscle, and/orjoint health in the subject, relative to prior to the administering. 29.A recombinant cell containing therein at least one copy of aheterologous nucleic acid sequence encoding a non-naturally occurringpolypeptide, wherein the non-naturally occurring polypeptide comprisesan amino acid sequence having at least 80% sequence identity to atruncate of SEQ ID NO: 32, wherein the truncate of SEQ ID NO: 32 has anN-terminal truncation of 50 amino acids to 750 amino acids, a C-terminaltruncation of 50 amino acids to 650 amino acids, or both the N-terminaltruncation and the C-terminal truncation, relative to SEQ ID NO:
 32. 30.The recombinant cell of claim 29, wherein the recombinant cell lacks anenzyme that hydroxylates one or more amino acids of the non-naturallyoccurring polypeptide, wherein the enzyme is selected from the groupconsisting of: prolyl 4-hydroxylase, prolyl 3-hydroxylase, and both.